Crystal structure of the large ribosomal subunit from S. aureus

ABSTRACT

A composition-of-matter comprising a crystallized form of a large ribosomal ( 50 S) subunit of a pathogenic bacterium, and the atomic coordinates of the three-dimensional structure thereof are provided herein, as well as methods for crystallizing the same, and using the atomic coordinates of the same to design de novo ligands with high specificity thereto.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/547,499 filed on Jul. 30, 2017, which is a National Phase of PCTPatent Application No. PCT/IL2016/050082 having International FilingDate of Jan. 26, 2016, which claims the benefit of priority under 35 USC§ 119(e) of U.S. Provisional Patent Application Ser. Nos. 62/109,185filed on Jan. 29, 2015, and 62/280,231 filed on Jan. 19, 2016. Thecontents of the above applications are all incorporated by reference asif fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 80470SequenceListing.txt, created on Dec. 17,2019, comprising 36,162 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference. The sequencelisting submitted herewith is identical to the sequence listing formingpart of the international application.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to crystalstructures and structure-based drug design and, more particularly, butnot exclusively, to methods of designing species-specific antimicrobialagents based on crystal structures of pathogenic ribosomal subunits.

The clinical usage of the currently available antibiotics is becomingever more limited due to the capability of pathogens to undergomutations, the phenotype thereof minimizes or abolishes bindinginteraction of the antibiotics to the molecular target in the pathogen.The emergence of bacterial resistance to antibiotics threatensregression to the pre-antibiotic era as the treatments of infectionswith the available arsenal of clinically used antibiotics have sufferedfrom the appearance of multidrug-resistant strains. Consequently, manyhospital-acquired infections are currently caused by highly resistantbacteria such as methicillin-resistant Staphylococcus aureus (MRSA) andvancomycin-resistant Staphylococcus aureus (VRSA), Gram positiveversatile and aggressive pathogens that are among the most worrisomepathogenic bacteria.

Among the existing antibiotics, many target the fundamental process ofprotein biosynthesis, mostly by interrupting bacteria's ribosomalactivity. A large number of antibiotics target the universal cellularmulti-components RNP (RNA-protein) particles translating the geneticcode into proteins.

The increasing epidemiology of Staphylococcus aureus (SA) infectionsrevealed that most of SA strains have 5-6 copies of the operon of themain rRNA (ribosomal RNA) component, namely the 23S rRNA. The fact thatSA has 5-6 operon copies is most likely correlated with the finding thatresistance that is caused by a single RNA mutation is accumulative.

It has been observed that in SA, the commonly occurring resistance toribosomal-active antibiotics is acquired by a single-nucleotide mutationin the 23S large ribosomal subunit rRNA. SA resistant mutations are alsoassociated with the region of rProtein (ribosomal protein) L3 that islocated in proximity to the peptidyl transferase center (PTC) andrProteins L4 and L22 that reach the exit tunnel.

Infections caused by SA are typically treated by several antibiotics,including the ribosomal-active antibiotics linezolid, telithromycin andpleuromutilins such as BC-3205, the structures of which are presentedhereinbelow, which bind to the large ribosomal subunit.

Linezolid is a synthetic drug which has been approved by FDA on April2000 for treating Gram positive pathogen infections. It belongs to theclass of oxazolidinones and was designed to bind at the PTC. Sincelinezolid is a synthetic drug, no pre-existing resistance mechanismswere known prior to its use, although resistance to otherribosomal-active antibiotics that target the same site has beenidentified. However, it was expected that emergence of resistance tothis drug would occur rather slowly. Despite these expectations, S.aureus linezolid resistance, acquired by a specific 23S point mutation,referred to as G2576U (E. coli numbering is used throughout), wasreported a year after its approval for treatment. Since linezolid isoften used as the last line of defense against multidrug-resistantbacterial infections, a resistance to linezolid in clinical isolates wasreported to be rather rare, and on 2010, it was reported to occur inless than 1% of SA isolates.

The observed G2576U resistance mutation is in accord with the crystalstructures and with a model of ribosome-linezolid complexes [Wilson, D.N. et al., P.N.A.S. USA, 2008, 105(36), p. 13339-44; Ippolito, J. A. etal., J Med Chem, 2008, 51(12), p. 3353-6; and Leach, K. L. et al.,Molecular Cell, 2007, 26(3), p. 393-402], which indicate that the drugbinding site is composed of 23S rRNA nucleotides that form the innershell of the PTC, thus perturbing the correct positioning of tRNAs onthe ribosome. Interestingly, the G2576U resistance mutation was found tobe associated with linezolid resistance in the multi-drug resistantbacteria Enterococcus faecium and Enterococcus faecalis and in S.pneumonia, as well as the tuberculosis diagnostic tool Mycobacteriumsmegmatis.

Telithromycin is a ketolide antibacterial agent/drug (antibiotic) thatis structurally related to macrolides, and which has been developedspecifically to provide optimal therapy for the treatment of respiratorytract infections (RTI) caused by either typical or atypical respiratorypathogens. Ketolides possess two innovative structural modifications, a3-keto group and a large N-substituted C11, C12-carbamate side chain.Telithromycin has an additional long alkyl-aryl arm. Telithromycin haveshowed potent in vitro activity against S. pneumoniae, including strainsresistant to macrolide-lincosamide-streptogramin B ketolide (MLS_(B)K)and sub-inhibitory concentrations of telithromycin have been found toinhibit MRSA in vitro.

Crystal structures showed that telithromycin binds to the largeribosomal subunit at the macrolide binding pocket in the ribosome's exittunnel [Berisio, R. et al., J Bacteriol., 2003, 185(14), p. 4276-9; Tu,D et al., Cell, 2005, 121(2), p. 257-70; Dunkle, J. A. et al., Proc NatlAcad Sci USA, 2010, 107(40), p. 17152-17157; and Bulkley, D. et al.,Proc Natl Acad Sci USA, 2010, 107(40), p. 17158-63], and that itsflexible alkyl-aryl arm is pointing in different directions in differentspecies.

Pleuromutilin and its derivatives are antibacterial agents (antibiotics)that inhibit protein synthesis in bacteria by binding to the peptidyltransferase component (PTC) of the 50S subunit of ribosomes.Pleuromutilin is a natural product of the fungi Pleurotus mutilus (alsoknown as Clitopilus scyphoides), which has been used as a base for thesynthesis of several semi-synthetic antibacterial agents, designed forclinical utilization by targeting eubacterial ribosomes. Members of thisclass of antibiotics, collectively referred to herein and in the art asPleuromutilins, which includes retapamulin, valnemulin and tiamulin andsome investigational drugs such as azamulin and BC-3781, all exhibit atricyclic mutilin core, a C21 keto group, essential for antimicrobialactivity, and various substituents at the C14, most of which areextensions of diverse chemical nature. Some of these semi-syntheticantibacterial agents are already in clinical use and exhibit elevatedactivity over a broad spectrum of pathogens.

Retapamulin belongs to the group of C14-sulfanyl-acetate derivatives ofPleuromutilin, and was approved for use as a topical antibiotic on 2007.Retapamulin was shown to possess potent activity against Gram positivepathogens, and a low propensity to develop resistance. Thus, all strainsof Staphylococcus aureus and Streptococcus pyogenes were susceptible toretapamulin at a minimal inhibition concentration (MIC) of 0.5 gram/ml.Other C14-sulfanyl-acetate derivatives of Pleuromutilin, valnemulin andtiamulin, were approved for veterinary clinical use.

Recent advances in pleuromutilin's chemistry yielded several newcompounds as potential antibacterial agents. Among them are BC-3205,which is a semi-synthetic pleuromutilin derivative that was developedfor oral treatment of skin and skin structure infections (SSSI) andcommunity-acquired pneumonia (CAP), as well as BC-3781 and BC-7013, allof which by Nabriva Therapeutics AG, Vienna, Austria. BC-3205 actsagainst SA with a MIC of 0.06 μg/ml, is 16-32-fold more potent thanlinezolid against SA and is therefore considered as highly potentantibacterial agent.

The available crystal structures of complexes of the large ribosomalsubunit from a non-pathogenic model bacterium, D50S, with variouspleuromutilin compounds, namely tiamulin, retapamulin, SB-264128 andSB-571519, revealed that these compounds are bound to the largeribosomal subunit at the PTC. In all cases the cores of these compoundsare placed in a similar fashion at the A-site, and the C14 extensions ofthese compounds are pointing towards the P-site, thus, directlyinhibiting peptide bond formation. As the PTC is almost fully conserved,the pleuromutilin's efficient inhibitory modes are attained byexploiting the ribosomal intrinsic functional flexibility forinduced-fit and remote conformational rearrangements that result intightening up the binding pockets [Schlünzen, F. et al., Molecularmicrobiology, 2004, 54, p. 1287-1294; and Davidovich, C. et al.,Proceedings of the National Academy of Sciences, 104, p. 4291-4296].

Species specificity in relevance to emergence of resistance toantibiotics in bacteria has been observed; however, so far all availablestructural information on ribosomal-antibiotics interactions has beenobtained from ribosomal particles and subunits of non-pathogenicbacteria, which only emphasized the common traits. Previous studieswhich compared structures of ribosomes from different kingdoms of life[Petrov, A.S. et al., P.N.A.S. USA, 2014, 111(28), p. 10251-10256] haveprompted suggestions concerning pathways in ribosome evolution.

Previous structural studies on antibiotics' modes of binding andbioactivity were based only on the available ribosomal crystalstructures, which were of eubacteria suitable to mimic pathogens underclinical relevant conditions. These include D50S of Deinococcusradiodurans [Schluenzen, F. et al., Nature, 2001, 413, p. 814-21], T70Sof Thermus thermophilus [Voorhees, R. M. et al., Nat Struct Mol Biol.,2009, 16(5), p. 528-33] and E70S from the non-pathogenic strain ofEscherichia coli [Schuwirth, B. S. et al., Science, 2005, 310(5749), p.827-834]. Results of these studies provided useful insights for commontraits of the mode of action of antibiotics, such as binding atribosomal functional sites, e.g. the PTC or the protein exit tunnel;illuminated structural bases for the distinction between patients(eukaryotes) and pathogens (eubacteria) despite the high conservation ofthe ribosomal functional sites; and shed light on antibiotics synergismand the general principles of resistance and cross resistance.

Based on the similarity in their sequences, the structures of ribosomesfrom pathogens are expected to resemble ribosomes from other eubacteria;however, species specificity in clinically relevant properties,particularly in the modes of acquiring antibiotic resistance, has beenidentified [Wilson, D. N., Ann. N. E Acad. Sci., 2011, 1241, p. 1-16],and it has been shown that small structural differences betweenbacterial species could affect the drug binding [Yonath, A., Mol Cells,2005, 20, p. 1-16].

Additional background art includes U.S. Pat. Nos. 6,638,908, 6,845,328,6,925,394, 6,939,848, 6,947,844, 6,947,845, 6,952,650, 7,079,956,7,133,783, 7,504,486, 7,606,670, 7,666,849 and 8,470,990; U.S. PatentApplication Publication Nos. 20020072861, 20020086308, 20020106660,20030027315, 20030099955, 20030153002, 20030171327, 20030232779,20040034207, 20040265984, 20050036997, 20050154538, 20050233349,20050234227, 20050272681, 20060136146, 20080057494, 20090081697,20100131258, 20100204253, 20100312525, 20120316106 and 20140066623; andInternational Patent Application Publication No. WO 2011/080739, WO2000/0693912 and WO 2003/026562.

SUMMARY OF THE INVENTION

The possession of the 3D-structure in atomic resolution of one of themost pivotal biomolecular targets, such as the ribosome of a pathogenicbacterium, opens the path to the design of highly specific and effectivede novo ligands which can used as drugs that inhibit protein synthesisof a pathogenic bacterium, such as Staphylococcus aureus, and thereforecan be used to treat medical conditions associated therewith.

According to an aspect of some embodiments of the present inventionthere is provided a composition-of-matter which includes a crystallizedlarge ribosomal subunit of a pathogenic bacterium, wherein thepathogenic bacterium is:

a pathogenic Gram positive bacterium; and/or

a pathogenic bacterium exhibiting a degree of 23S rRNA sequence identityof at least 80% compared to rRNA of Staphylococcus aureus; and/or

a pathogenic bacterium exhibiting a degree of 23S rRNA sequence identityof less than 99.9% compared to rRNA of Escherichia coli, and

the crystallized large ribosomal subunit effectively diffracts X-raysfor calculating an electron density map and determination of atomiccoordinates to a resolution of at least 4 Å.

According to some embodiments of the invention, the Gram positivepathogenic bacterium is a Gram positive cocci bacterium.

According to some embodiments of the invention, the Gram positive coccibacterium is a Staphylococcus bacterium.

According to some embodiments of the invention, the Staphylococcusbacterium is Staphylococcus aureus.

According to some embodiments of the invention, Staphylococcus aureus iscapable of developing a resistance to an antibacterial agent.

According to some embodiments of the invention, the Staphylococcusaureus is selected from the group consisting of a methicillin-resistantStaphylococcus aureus (MRSA), an oxacillin-resistant Staphylococcusaureus (ORSA), a vancomycin-resistant Staphylococcus aureus (VRSA) and avancomycin intermediate Staphylococcus aureus (VISA).

According to some of the respective embodiments of the invention, thecrystallized large ribosomal subunit is characterized by a crystal spacegroup of P6522 and a unit cell dimensions of a=279.8±10 Å, b=279.8±10 Å,c=872.8±10 Å, α=90, β=90 and γ=120.

According to some of the respective embodiments of the invention, thecrystallized large ribosomal subunit is characterized by the atomiccoordinates deposited at the Protein Data Bank under accession numberPDB ID: 4WCE.

According to some of any of the embodiments of the invention, a ligandis bound to the large ribosomal subunit to thereby form a crystallizedcomplex of the subunit and the ligand.

According to some of the respective embodiments of the invention, theligand is an antibacterial agent. According to some embodiments of theinvention, the ligand is linezolid.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by a crystal space group of P6522and a unit cell dimensions of a=279.9±10 Å, b=279.9±10 Å, c=870.6±10 Å,α=90, β=90 and γ=120.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by the atomic coordinatesdeposited at the Protein Data Bank under accession number PDB ID: 4WFA.

According to some embodiments of the invention, the ligand is BC-3205.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by a crystal space group of P6522and a unit cell dimensions of a=280.9±10 Å, b=280.9±10 Å, c=875.6±10 Å,α=90, β=90 and γ=120.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by the atomic coordinatesdeposited at the Protein Data Bank under accession number PDB ID: 4WFB.

According to some embodiments of the invention, the ligand istelithromycin.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by a crystal space group of P6522and a unit cell dimensions of a=282.7±10 Å, b=282.7±10 Å, c=877.1±10 Å,α=90, β=90 and γ=120.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by the atomic coordinatesdeposited at the Protein Data Bank under accession number PDB ID: 4WF9.

According to some embodiments of the invention, the ligand is lefamulin.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by a crystal space group of P6522and a unit cell dimensions of a=282.1±10 Å, b=282.1±10 Å, c=875.3±10 Å,α=90, β=90 and γ=120.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by the atomic coordinatesdeposited at the Protein Data Bank under accession number PDB ID: 5HL7.

According to some embodiments of the invention, the ligand islincomycin.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by a crystal space group of P6522and a unit cell dimensions of a=280.8±10 Å, b=280.8±10 Å, c=873.5±10 Å,α=90, β=90 and γ=120.

According to some of the respective embodiments of the invention, thecrystallized complex is characterized by the atomic coordinatesdeposited at the Protein Data Bank under accession number PDB ID: 5HKV.

According to an aspect of some embodiments of the present inventionthere is provided a computer system which includes:

(a) a data-storage device having stored therein positioning dataindicative of atomic coordinates determined from an electron density maphaving a resolution of at least 4 Å calculated from X-rays diffractiondata obtained using at least one of any of the compositions-of-matterdescribed herein;

(b) a processing unit in electrical communication with the data-storagedevice and operating; and

(c) a program for calculating a three-dimensional model representativeof the large ribosomal subunit from the positioning data.

According to some embodiments of the invention, the computer systemfurther includes a device for providing a visual representation of themodel.

According to some of the respective embodiments of the invention, thepositioning data comprise at least a portion of the atomic coordinatesdeposited at the Protein Data Bank under accession numbers that includePDB IDs: 4WCE, 4WFA, 4WFB, 4WF9, 5HL7 and 5HKV.

According to some of any of the respective embodiments of the invention,the atomic coordinates define at least a portion of a ligand bound tothe large ribosomal subunit.

According to some of any embodiments of the invention, the ligand isselected from the group consisting of linezolid, BC-3205, telithromycin,lefamulin and lincomycin.

According to some of any of the respective embodiments of the invention,the positioning data comprise at least a portion of the atomiccoordinates deposited at the Protein Data Bank under accession numberselected from the group consisting of PDB IDs: 4WFA (linezolid), 4WFB(BC-3205) and 4WF9 (telithromycin), 5HL7 (lefamulin) and 5HKV(lincomycin).

According to some of any of the respective embodiments of the invention,the atomic coordinates are produced by molecular replacement using atleast a portion of the atomic coordinates of another large ribosomalsubunit.

According to some embodiments of the invention, the atomic coordinatesof the other large ribosomal subunit used for molecular replacement aredeposited at the Protein Data Bank under accession number PDB ID: 2ZJR.

According to some of any of the respective embodiments of the invention,the atomic coordinates comprise atomic coordinates of at least oneribosomal RNA (rRNA).

According to some of any of the respective embodiments of the invention,the atomic coordinates comprise atomic coordinates of at least oneribosomal protein (rProtein).

According to some of any of the respective embodiments of the invention,the atomic coordinates comprise at least a portion of at least onebinding site in the large ribosomal subunit.

According to some of any of the respective embodiments of the invention,the binding site is selected from the group consisting of aninter-subunit interface, a peptidyl transferase site, a GTPase center,an mRNA binding site, an A-site, a P-site, an E-site, a polypeptide exittunnel, a translation initiation factor (IF1) binding site, atranslation initiation factor (IF2) binding site, a translationinitiation factor (IF3) binding site, an elongation factor G (EF-G)binding site, elongation factor Tu (EF-Tu) binding site, hibernationfactor HPF binding site, hibernation factor RMF binding site,hibernation factor YfiA binding site, a GTP binding site and a ricinbinding site.

According to some of any of the respective embodiments of the invention,the atomic coordinates further comprise at least a portion of at leastone of the following, mRNA, tRNA, activated tRNA and a polypeptide undertranslation.

According to some embodiments of the invention, the computer systemfurther includes a computer-aided drug design program or computer-aideddrug design software suite for calculating and designing a structure ofa putative ligand for binding to one of the binding sites.

According to some of any of the respective embodiments of the invention,the computer system further includes atomic coordinates of a putativeligand for binding to the binding site in the large ribosomal subunit ofthe pathogenic bacterium, the computer-aided drug design is astructure-based drug design, and the putative ligand is designed basedon a structure of the binding site in the large ribosomal subunitprovided herein.

According to some embodiments of the invention, the computer systemfurther includes the data-storage device further stores:

(i) sequence data indicative of at least a portion of the amino acidsand at least a portion of the ribonucleic acids of the large ribosomalsubunit;

(ii) positioning data indicative of atomic coordinates of at least aportion of a large ribosomal subunit of at least one different organism;and

(iii) sequence data indicative of at least a portion of the amino acidsand at least a portion of the ribonucleic acids of the large ribosomalsubunit of the at least one different organism.

According to some embodiments of the invention, the computer systemfurther includes:

(d) a sequence alignment program for calculating a residue correlationdata of the amino acids and the ribonucleic acids of at least two largeribosomal subunits of at least two different organisms provided that atleast one of the at least two different organisms is the pathogenicbacterium; and

(e) a structure alignment program for superimposing the atomiccoordinates of the at least two large ribosomal subunits based on theresidue correlation data.

According to some of any of the respective embodiments of the invention,the different organism is selected from the group consisting of Thermusthermophilus, Escherichia coli, Haloarcula marismortui, Deinococcusradiodurans and Saccharomyces cerevisiae, Tetrahymena thermophila.

According to some of any of the respective embodiments of the invention,the atomic coordinates of the D. radiodurans comprise at least a portionof the atomic coordinates deposited at the Protein Data Bank underaccession number selected from the group consisting of PDB IDs: 2ZJR,3DLL, 2OGM, 2OGN, 2OGO, 1XNP, 1SM1, 1P9X, 1JZX, 1JZY and 4U67.

According to some of any of the respective embodiments of the invention,the atomic coordinates of the E. coli comprise at least a portion of theatomic coordinates deposited at the Protein Data Bank under accessionnumber selected from the group consisting of PDB IDs: 2AW4, 3R8S, 3OAT,30FZ and 3OFR.

According to some of any of the respective embodiments of the invention,the atomic coordinates of the T. thermophilus comprise at least aportion of the atomic coordinates deposited at the Protein Data Bankunder accession number selected from the group consisting of PDB IDs:2WDL, 2WDK, 3OI3 and 3OHD.

According to some of any of the respective embodiments of the invention,the atomic coordinates of the H. marismortui comprise at least a portionof the atomic coordinates deposited at the Protein Data Bank underaccession number selected from the group consisting of PDB ID: 1S72,3CC2 3CPW, 1YJN and 1YIJ.

According to some of any of the respective embodiments of the invention,the atomic coordinates of the T. thermophila comprise at least a portionof the atomic coordinates deposited at the Protein Data Bank underaccession number selected from the group consisting of PDB ID: 4A17,4A18, 4A19, 4A1A, 4A1B, 4A1C, 4A1D and 4A1E.

According to some of any of the respective embodiments of the invention,the atomic coordinates of the S. cerevisiae comprise at least a portionof the atomic coordinates deposited at the Protein Data Bank underaccession number selected from the group consisting of PDB ID: 3U5B,3U5C, 3U5D, 3U5E, 3U5F, 3U5G, 3U5H and 3U5I.

According to some of any of the respective embodiments of the invention,the different organism is a host of the pathogenic bacterium, therebyallowing calculating positioning data indicative of atomic coordinatesof the large ribosomal subunit of the host organism based on the residuecorrelation data.

According to some of any of the respective embodiments of the invention,the host organism is a mammal and the pathogenic bacterium is a pathogenof the mammal.

According to some of any of the respective embodiments of the invention,the mammal is a human.

According to some of any of the respective embodiments of the invention,the data-storage device further includes at least a portion of theatomic coordinates of a ribosomal subunit of a human.

According to some of any of the respective embodiments of the invention,the atomic coordinates of a ribosomal subunit of a human are afforded bya method selected from the group consisting of X-ray crystallography,NMR, molecular modeling, single-particle cryogenic electron microscopyand X-ray free-electron laser.

According to some of any of the respective embodiments of the invention,the computer system further includes atomic coordinates of aspecies-selective putative ligand for binding to the binding site in thelarge ribosomal subunit of the pathogenic bacterium, the computer-aideddrug design is a structure-based drug design, and the putative ligand isdesigned based on a structure of the binding site in the large ribosomalsubunit provided herein.

According to an aspect of some embodiments of the present inventionthere is provided a computer readable medium which includes retrievablepositioning data indicative of atomic coordinates determined from anelectron density map having a resolution of at least 4 Å calculated fromX-rays diffraction data obtained using the composition-of-matteraccording to any of the respective embodiments of the present invention.

According to some embodiments of the invention, the atomic coordinatesdeposited at the Protein Data Bank under accession number selected fromthe group consisting of PDB IDs: 4WCE, 4WFA, 4WFB, 4WF9, 5HL7 and 5HKV.

According to some embodiments of the invention, the computer readablemedium further includes atomic coordinates of at least a portion of alarge ribosomal subunit of D. radiodurans deposited at the Protein DataBank under accession number selected from the group consisting of PDBIDs: 2ZJR, 3DLL, 2OGM, 2OGN, 2OGO, 1XNP, 1SM1, 1P9X, 1JZX, 1JZY and4U67.

According to some embodiments of the invention, the computer readablemedium further includes atomic coordinates of at least a portion of alarge ribosomal subunit of E. coli deposited at the Protein Data Bankunder accession number selected from the group consisting of PDB IDs:2AW4, 3R8S, 3OAT, 30FZ and 3OFR.

According to some embodiments of the invention, the computer readablemedium further includes atomic coordinates of at least a portion of alarge ribosomal subunit of T. thermophilus deposited at the Protein DataBank under accession number selected from the group consisting of PDBIDs: 2WDL, 2WDK, 3OI3 and 3OHD.

According to some embodiments of the invention, the computer readablemedium further includes atomic coordinates of at least a portion of alarge ribosomal subunit of H. marismortui deposited at the Protein DataBank under accession number selected from the group consisting of PDBIDs: 1S72, 3CC2 3CPW, 1YJN and 1YIJ.

According to some embodiments of the invention, the computer readablemedium further includes atomic coordinates of at least a portion of alarge ribosomal subunit of T. thermophila deposited at the Protein DataBank under accession number selected from the group consisting of PDBIDs: 4A17, 4A18, 4A19, 4A1A, 4A1B, 4A1C, 4A1D and 4A1E.

According to some embodiments of the invention, the computer readablemedium further includes atomic coordinates of at least a portion of alarge ribosomal subunit of S. cerevisiae deposited at the Protein DataBank under accession number selected from the group consisting of PDBIDs: 3U5B, 3U5C, 3U5D, 3U5E, 3U5F, 3U5G, 3U5H and 3U5I.

According to some embodiments of the invention, the computer readablemedium further includes atomic coordinates of at least a portion of alarge ribosomal subunit of a human afforded by a method selected fromthe group consisting of molecular modeling, single-particle cryogenicelectron microscopy and X-ray free-electron laser.

According to some of any of the respective embodiments of the invention,the computer readable medium further includes sequence data indicativeof at least a portion of the amino acids and at least a portion of theribonucleic acids of the large ribosomal subunit.

According to some of any of the respective embodiments of the invention,the computer readable medium further includes sequence data indicativeof at least a portion of the amino acids and at least a portion of theribonucleic acids of a large ribosomal subunit of a host organism.

According to some of any of the respective embodiments of the invention,the computer readable medium further includes positioning dataindicative of atomic coordinates of the large ribosomal subunit of thehost organism.

According to some of the respective embodiments of the invention, thehost organism is a mammal and the pathogenic bacterium is a pathogen ofthe mammal.

According to some of the respective embodiments of the invention, themammal is a human.

According to an aspect of some embodiments of the present inventionthere is provided a method for designing a putative ligand having anaffinity to a binding site of a large ribosomal subunit of a pathogenicbacterium, the method is effected by:

(a) obtaining positioning data indicative of atomic coordinates of atleast one binding site determined from an electron density map having aresolution of at least 4 Å calculated from X-rays diffraction dataobtained using at least one of the composition-of-matter according toany of the respective embodiments of the present invention;

(b) calculating a molecular surface of the binding site; and

(c) computationally constructing a chemically feasible ligand having amolecular surface that match the molecular surface of the binding site.

According to some embodiments of the invention, the method furtherincludes, prior to step (b), determining the binding site in the largeribosomal subunit using the positioning data (atomic coordinates) of atleast a portion of a ligand bound to the large ribosomal subunit,wherein the binding site being in association with the ligand (atarget-bound ligand).

According to some embodiments of the invention, the method furtherincludes, prior to step (c): computationally constructing a library ofstructures of chemically feasible ligands having a molecular surfacethat matches the molecular surface of the binding site.

According to some embodiments of the invention, the method furtherincludes:

(d) computationally determining a matching score for each of theligands; and

(e) based on the matching score selecting at least one putative ligandhaving the desired affinity to the binding site of the large ribosomalsubunit of a pathogenic bacterium.

According to some embodiments of the invention, the method furtherincludes, prior to step (d), adding to the library a plurality ofstructures of chemically feasible variants of pre-existing ligands.

According to some embodiments of the invention, the method furtherincludes, prior to step (c), calculating a molecular surface of at leasta portion of the binding site of a large ribosomal subunit of adifferent organism.

According to some of any of the respective embodiments of the invention,the different organism used in the method for designing a putativeligand, is selected from the group consisting of a host of thepathogenic bacterium and a benign microorganism.

According to some embodiments of the invention, step (c) of the methodfurther includes computationally constructing a chemically feasibleligand having a molecular surface that matches the molecular surface ofthe binding site of the large ribosomal subunit of a pathogenicbacterium, and mismatches at least one feature in the molecular surfaceof the binding site in the of a large ribosomal subunit of the differentorganism.

According to some of the respective embodiments of the invention, theactive site considered in the method is selected from the groupconsisting of a inter-subunit interface, a peptidyl transferase site, aGTPase center, an mRNA binding site, an A-site, a P-site, an E-site, apolypeptide exit tunnel, a translation initiation factor (IF1) bindingsite, a translation initiation factor (IF2) binding site, a translationinitiation factor (IF3) binding site, an elongation factor G (EF-G)binding site, elongation factor Tu (EF-Tu) binding site, hibernationfactor HPF binding site, hibernation factor RMF binding site,hibernation factor YfiA binding site, a GTP binding site and a ricinbinding site.

According to some of any of the respective embodiments of the invention,the ligand has a molecular weight of less than 1,500 g/mol.

According to some of any of the respective embodiments of the invention,the method further includes, subsequent to step (c), preparing (e.g.,synthesizing) the ligand.

According to some of any of the respective embodiments of the invention,the method further includes contacting the ligand with the largeribosomal subunit of the pathogenic bacterium.

In some embodiments of the invention, contacting the ligand with thelarge ribosomal subunit is effected in an activity assay which alsoproduces a binding score.

According to an aspect of some embodiments of the present inventionthere is provided a ligand having an affinity to a molecular surface ofat least a portion of a binding site of a large ribosomal subunit of apathogenic bacterium designed by the method for designing a putativeligand, according to any of the respective embodiments of the presentinvention.

According to some embodiments of the invention, the ligand is a proteinsynthesis inhibitor.

According to some embodiments of the invention, the ligand is a proteinsynthesis inhibitor that includes:

a first binding moiety having a molecular surface that mimics orduplicates a molecular surface of a first pre-existing ligand that bindsto a first binding site in a large ribosomal subunit; and

at least one second binding moiety having a molecular surface thatmimics or duplicates a surface of a second pre-existing ligand thatbinds with a second binding site in the ribosomal subunit,

wherein:

the first pre-existing ligand is different than the second pre-existingligand;

the first binding site is different than the second binding site;

the first binding moiety is attached to the second binding moiety via alinking moiety so as to permit both the first moiety and the secondmoiety to bind simultaneously each with its respective binding sitethereby disrupting protein synthesis in a ribosomal subunit.

According to some of the respective embodiments of the invention, theprotein synthesis inhibitor has a molecular weight of less than about1,500 g/mol.

According to some of the respective embodiments of the invention, theaffinity constant of the ligand with respect to the ribosomal subunit isgreater than each of the affinity constants of the first pre-existingligand and the second pre-existing ligand.

According to some of the respective embodiments of the invention, thepre-existing ligand is an antibacterial agent.

According to some of the respective embodiments of the invention, eachof the first pre-existing ligand and the second pre-existing ligand isselected from the group consisting of linezolid, BC-3205, telithromycin,lefamulin and lincomycin.

According to an aspect of some embodiments of the present inventionthere is provided a ligand which includes:

a first binding moiety having a molecular surface that mimics orduplicates a molecular surface of a first pre-existing ligand that bindsto a first binding site in a large ribosomal subunit; and

a second binding moiety having a molecular surface that mimics orduplicates a surface of a second pre-existing ligand that binds with asecond binding site in the ribosomal subunit,

wherein:

the first pre-existing ligand is not the second pre-existing ligand;

the first binding site is not the second binding site;

the first binding moiety is attached to the second binding moiety via alinking moiety so as to permit both the first moiety and the secondmoiety to bind simultaneously each with its respective binding sitethereby disrupting protein synthesis in a ribosomal subunit, wherein apositioning data of the first binding moiety relative to a positioningdata of the second binding moiety, is determined according to atomiccoordinates indicative of a positioning data of the first binding moietyand the second binding moiety obtained from an electron density maphaving a resolution of at least 4 Å calculated from X-rays diffractiondata obtained using at least one of the composition-of-matter accordingto any of the respective embodiments of the present invention.

According to some of the respective embodiments of the invention, theprotein synthesis inhibitor has a molecular weight of less than about1,500 g/mol.

According to some of the respective embodiments of the invention, theaffinity constant of the ligand with respect to the ribosomal subunit isgreater than each of an affinity constant of the first pre-existingligand and the second pre-existing ligand.

According to some of the respective embodiments of the invention, thepre-existing ligand is an antibacterial agent.

According to some of the respective embodiments of the invention, eachof the first pre-existing ligand and the second pre-existing ligand isselected from the group consisting of linezolid, BC-3205, telithromycin,lefamulin and lincomycin.

According to some of the respective embodiments of the invention, theribosomal subunit is a large ribosomal subunit of a pathogenic Grampositive bacterium.

According to some of the respective embodiments of the invention, theligand is a pathogenic Gram positive bacterium-specific proteinsynthesis inhibitor.

According to some of the respective embodiments of the invention, thepathogenic Gram positive bacterium is Staphylococcus aureus.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition which includes, as anactive ingredient, the ligand according to any of the respectiveembodiments of the invention, and a pharmaceutically acceptable carrier.

According to some embodiments of the invention, the pharmaceuticalcomposition is packaged in a packaging material and identified in print,in or on the packaging material, for use in the treatment of aninfection associated with a pathogenic bacterium, wherein the pathogenicbacterium is Gram positive and/or exhibiting a degree of 23S rRNAsequence identity of at least 80% compared to rRNA of Staphylococcusaureus and a degree of 23S rRNA sequence identity of less than 99.9%compared to rRNA of Escherichia coli.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating an infection associated with apathogenic bacterium, the method is effected by administering to asubject in need thereof, e.g., a human, a therapeutically effectiveamount of a ligand according to any of the respective embodiments of thepresent invention, wherein the pathogenic bacterium is Gram positiveand/or exhibiting a degree of 23S rRNA sequence identity of at least 80%compared to rRNA of Staphylococcus aureus and a degree of 23S rRNAsequence identity of less than 99.9% compared to rRNA of Escherichiacoli.

According to an aspect of some embodiments of the present inventionthere is provided a method of obtaining the composition-of-matteraccording to any of the respective embodiments of the present invention,the method is effected by:

(a) subjecting a crystallization solution that includes a concentratedpurified preparation of the large ribosomal subunit of the pathogenicbacterium to vapor diffusion conditions against a reservoir solution fora first time period;

(e) transferring at least one macro crystal to a stabilization solution,to thereby obtain a crystal of the large ribosomal subunit of thepathogenic bacterium capable of diffracting X-ray to resolution of atleast 4 Å.

According to some embodiments of the present invention, the method ofobtaining the composition-of-matter further includes, prior to step (e):

(b) extracting a plurality of seeding crystals from the crystallizationsolution;

(c) transferring at least one of the seeding crystals into thecrystallization solution; and

(d) subjecting the crystallization solution that includes the seedingcrystals to vapor diffusion conditions against the reservoir solutionfor a second time period.

According to some embodiments of the present invention, the concentratedpurified preparation is prepared by concentrating a fraction of a zonalultracentrifugation using a sucrose gradient.

According to some of any of the respective embodiments of the presentinvention, the concentrated purified preparation is characterized by anoptical absorbance at 260 nm (A260) of 600-1000 per ml.

According to some of any of the respective embodiments of the presentinvention, vapor diffusion conditions include a drop of thecrystallization solution applied substantially at the center and bottomof an air-tight lid that closes a top opening of a container whichincludes the reservoir solution.

According to some of any of the respective embodiments of the presentinvention, the crystallization solution comprises the concentratedpurified preparation at a concentration of 0.9-1.6 mg/ml, 0.1-0.2percent by weight 2-methyl-2,4-pentanediol, 0.3-0.4 percent by weightethanol, 4-6 mM spermidine, 0.4-0.6 mM MnCl₂, 20 mM Hepes buffer set topH range of 6.8-7.8, 10 mM MgCl₂, 60 mM NH₄Cl and 15 mM KCl.

According to some of any of the respective embodiments of the presentinvention, the reservoir solution comprises 4-6 percent by weight2-methyl-2,4-pentanediol, 8-12 percent by weight ethanol, 110 mM Hepesbuffer set to pH range of 6.8-7.8, 10 mM MgCl₂, 60 mM NH₄Cl and 15 mMKCl.

According to some of any of the respective embodiments of the presentinvention, the stabilization solution comprises 10-25 percent by weight2-methyl-2,4-pentanediol, 10-20 percent by weight ethanol, 110 mM Hepesbuffer set to pH range of 6.8-7.8, 10 mM MgCl₂, 60 mM NH₄Cl and 15 mMKCl.

According to some embodiments of the present invention, step (c) of themethod of obtaining the composition-of-matter, if present, furtherincludes, prior to the transferring, washing the seeding crystals in asolution that includes 7-8 percent by weight 2-methyl-2,4-pentanediol,7-8 percent by weight ethanol, 110 mM Hepes buffer set to pH range of6.8-7.8, 10 mM MgCl₂, 60 mM NH₄Cl and 15 mM KCl buffer and 0.5 mM MnCl₂.

According to some embodiments of the present invention, the method ofobtaining the composition-of-matter further includes, prior to step (a),heating the concentrated purified preparation of the large ribosomalsubunit for 20-40 minutes at 37° C.

According to some embodiments of the present invention, the first timeperiod ranges from 10 days to 20 days.

According to some embodiments of the present invention, the second timeperiod, if present, ranges from 2 weeks to 4 weeks.

According to some embodiments of the present invention, each of any oneof steps (a)-(e) of the method of obtaining the composition-of-matter,if present, is effected at a temperature that ranges from 18° C. to 22°C.

According to some embodiments of the present invention, the method ofobtaining the composition-of-matter further comprises:

(f) transferring at least one macro crystal from the stabilizationsolution to a soaking solution that includes a ligand dissolved in thestabilization solution; and

(g) maintaining the crystal in the soaking solution for a third timeperiod that ranges from 1 day to 10 days.

According to some embodiments of the present invention, the third timeperiod ranges from 2 hours to 24 hours prior to exposing the crystalhaving the ligand soaked therein to X-ray.

According to some embodiments of the present invention, theconcentration of the ligand in the soaking solution ranges from 1 μg/mlto 30 μg/ml.

According to some of any of the respective embodiments of the presentinvention, the ligand is an antibacterial agent.

According to some of any of the respective embodiments of the presentinvention, the antibacterial agent is selected from the group consistingof linezolid, BC-3205 and telithromycin.

According to an aspect of some embodiments of the present inventionthere is provided a crystal of a large ribosomal subunit of a pathogenicbacterium produced by the method according to any of the respectiveembodiments of the invention.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

Implementation of the method and/or system of embodiments of theinvention can involve performing or completing selected tasks manually,automatically, or a combination thereof. Moreover, according to actualinstrumentation and equipment of embodiments of the method and/or systemof the invention, several selected tasks could be implemented byhardware, by software or by firmware or by a combination thereof usingan operating system.

For example, computer systems or otherwise any hardware for performingselected tasks according to embodiments of the invention could beimplemented as a chip or a circuit. As software, selected tasksaccording to embodiments of the invention could be implemented as aplurality of software instructions being executed by a computer usingany suitable operating system. In an exemplary embodiment of theinvention, one or more tasks according to exemplary embodiments ofmethod and/or system as described herein are performed by a dataprocessor, such as a computing platform or system for executing aplurality of instructions. Optionally, the data processor includes avolatile memory for storing instructions and/or data and/or anon-volatile storage, for example, a magnetic hard-disk and/or removablemedia, for storing instructions and/or data. Optionally, a networkconnection is provided as well. A display and/or a user input devicesuch as a keyboard or mouse are optionally provided as well.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 presents a graphic illustration of the crystal structure of thelarge ribosomal subunit of SA (SA50S), according to some embodiments ofthe present invention, wherein the rRNA is colored in grey, therProteins are colored in various colors, the PTC is marked by a red starand the approximate path of the internal exit tunnel is marked by a bandcolored in dark blue;

FIGS. 2A-2F present the weighted 2F_(o)-F_(c) electron density maps oflinezolid (FIG. 2A), telithromycin (FIG. 2B) and BC-3205 (FIG. 2C),contoured at 1.0 σ, and the weighted F_(o)-F_(c) electron density mapsof linezolid (FIG. 2D), telithromycin (FIG. 2E) and BC-3205 (FIG. 2F),contoured at 3.0 σ;

FIGS. 3A-3B present a graphic illustration of the structure of SA50S,showing relative locations of the rRNA regions with fold variability onthe SA50S subunit, wherein SA50S 23S rRNA is shown in teal, and thevariable regions are shown in orange, whereby (FIG. 3A) and (FIG. 3B)are rotated 90° with respect to each other;

FIGS. 4A-4H present graphic illustrations of superimposed structuremodels of S. aureus (colored in teal), D. radiodurans (colored in grey),E. coli (colored in purple) and T. thermophilus (colored in orange),showing the structural variability in the rRNA backbone, wherein in FIG.4A the h25 region is emphasized, in FIG. 4B the h9 region is emphasized,in FIG. 4C the h63 region is emphasized, in FIG. 4D the h10 region isemphasized, in FIG. 4E the h79 region is emphasized, in FIG. 4F the h15and h16 regions are emphasized, in FIG. 4G the h68 region is emphasized,and in FIG. 4H the h28 region is emphasized;

FIGS. 5A-5B present graphic illustrations of the flexible nucleotides atthe PTC and at the exit tunnel (FIG. 5A), showing U2506, U2585, A2062,A2602 and U2491, where the P-site tRNA (shown as a green surface) andthe A-site tRNA (shown as a blue surface) would bind, whereas S. aureus23S RNA backbone and nucleotides are colored in teal, and D.radiodurans, T. thermophilus and E. coli nucleotides are shown in grey,orange and purple, respectively, and further showing the flexiblenucleotides towards the tunnel opening (FIG. 5B), wherein A90, A91 andA508 are located in the ribosomal exit tunnel, detected with differentconformations in all four structures, and a possible path of thebackbone of a modeled nascent poly-alanine chain is represented by athick yellow string;

FIGS. 6A-6D present graphic illustrations of the surface of the SA50Sindicating the locations of the globular regions of the rProteins,whereas rRNA is shown in grey and the various rProteins are shown indifferent colors, showing a view from the SA50S intersubunit surface(FIG. 6A), a view from the SA50S outer surface (FIG. 6B), and views of a+90 degrees and −90 degrees vertical rotation of the intersubunitsurface (FIG. 6C and FIG. 6D, respectively);

FIGS. 7A-7B present graphic illustrations of the subunit interface,showing some of the structural differences in the rProteins foundbetween SA50S, T70S, E70S and D50S, while focusing on L5 (FIG. 7A) andL16 (FIG. 7B);

FIGS. 8A-8B present graphic illustrations emphasizing the structuraldifferences between SA50S, T70S, E70S and D50S in the rProteins thatinteract with substrates, while focusing on L28 (FIG. 8A) and L27 (FIG.8B);

FIGS. 9A-9G present graphic illustrations emphasizing the structuraldifferences between SA50S, T70S, E70S and D50S in the rProteins at therims of the erythromycin binding site (FIG. 9A), the exit tunnel openingfocusing on L23, L24, and L29 (FIG. 9B), L24 in all 4 structures (FIG.9C), the central protuberance (FIG. 9D); the L25, G11-L26, I49-T69,F79-186 loops, and the L16 C-terminal (FIG. 9E), the L25 and L16C-terminal (FIG. 9F), and the L27 R79-K85 loop and C-terminal fold (FIG.9G), whereas the possible path of the backbone of a modeled nascentprotein chain is indicated in lime/yellow in some of the illustrations;

FIGS. 10A-10H present graphic illustrations emphasizing the structuraldifferences between S. aureus (colored in teal), D. radiodurans (coloredin grey), T. thermophilus (colored in orange) and E. coli (colored inpurple) in some rProteins at the subunit surface, focusing on the L3A57-L67 loop (FIG. 10A), the L17 T65-A81 loop (FIG. 10B), the V6-117surface loop of n L4 and N-terminal of L15 (FIG. 10C), the C andN-terminals of L4 (FIGS. 10D-E), the L15 loops I69-T89 and T89-V97 (FIG.10F), the L15 N-terminal (FIG. 10G), and the L28 globular domain (FIG.10H);

FIGS. 11A-11B present graphic illustrations emphasizing the structuraldifferences in the N-terminal of protein L32 that resides in the secondshell around the erythromycin binding pocket, as seen in SA50S and E70S(FIG. 11A) and in T70S and D50S (FIG. 11B);

FIGS. 12A-12F present graphic illustrations of SA50S in complex withlinezolid, referred to herein as SA50Slin (FIG. 12A and FIG. 12B), SA50Sin complex with telithromycin, referred to herein as SA50Steli (FIG. 12Cand FIG. 12D) and SA50S in complex with BC-3205, referred to herein asSA50S-BC3205 (FIG. 12E and FIG. 12F);

FIGS. 13A-13F present graphic illustrations of structures of nativeSA50S rRNA and rProteins (colored in teal), D50S (colored in grey), E70S(colored in purple) and T70S (colored in orange) superimposed forcomparative analysis and study of the resistance and cross resistancemechanisms in SA, showing rRNA nucleotides of SA in regions where theycan be well aligned with the corresponding nucleotides in all otherstructures used for the comparisons, focusing on L11 (FIG. 13A), L3(FIG. 13B), the rRNA binding pocket (FIGS. 13C-D), and L4 (FIGS. 13E-F);

FIGS. 14A-14C present graphic illustrations of regions in thesuperimposed structures of S. cerevisiae 60S (PDB ID: 3U5D) (colored inyellow), T. thermophilia 60S (PDB ID: 4A18) (colored in red) and SA50S(colored in teal), showing the sequence and structural variability amongeukaryotes and prokaryotes rRNA antibiotics binding pockets and vicinitywhen erythromycin (FIG. 14A), telithromycin (FIG. 14B) and tiamulin(FIG. 14C) are bound;

FIG. 15 presents an illustration of linezolid (green stick model),BC-3205 (blue stick model) and telithromycin (red stick model), as thesethree ligands are positioned in the crystal structure of thecorresponding complex with SA50S, wherein each complex structures issuperimposed on the native SA50S crystal structure, and further presentsthe molecular surface of the combined ligand structures illustrated as awire mesh encasing the three ligands, wherein the coloring of meshcorresponds to the color of the ligand which contributes to themolecular surface at the corresponding region thereof;

FIG. 16 presents a graphic illustration of an overlay of lefamulin inthe pleouromutilin binding site, as elucidated from the complexstructures SA50Slef (orange), D50S-retapamulin (cyan; PDB ID: 2OGO) andD50S-SB280080 (lemon; PDB ID: 2OGN);

FIG. 17 presents a graphic illustration of an overlay of the PTC innative SA50S (teal) and in SA50Slef complex (orange), revealing themovements of nucleotide U2585 in the bound vs. native structure;

FIG. 18 presents a graphic illustration of an overlay of the PTC incomplex structure SA50SBC-3205 (magenta) and in complex structureSA50Slef (orange);

FIG. 19 presents a graphic illustration of the ribosomal binding pocketof lefamulin, as seen in the complex crystal structure SA50Slef, showingthat the ligand is held within the PTC by a net of hydrogen bonds withthe 23S rRNA, wherein the U-U interactions between U2585 and U2506stabilizes the lefamulin binding pocket (the electron density oflefamulin is weighted 2F_(o)-F_(c) contoured at 1.0 σ);

FIGS. 20A-20B present the results of in vitro transcription-translationcell-free inhibition assays of bacterial protein synthesis, wherein theinhibitory effect on protein expression in S. aureus system of BC-3205is presented in FIG. 20A and of lefamulin is presented in FIG. 20B. Theactivity of the reporter protein (luciferase) in the presence of variousconcentrations of BC-3205 and lefamulin is shown as arbitrary unit ofluminescence [a.u.]. The IC₅₀ values calculated by the plotted datashowed better inhibition of lefamulin than BC-3205 on protein synthesis;

FIG. 21 presents a graphic illustration of the electron density map(weighted 2Fo-Fc contoured at 1.0 σ) attributed to a molecule oflincomycin as seen in the crystal structure of the antibiotic agent incomplex with SA50S;

FIG. 22 presents a graphic illustration showing a structuralsuperposition of the two lincosamides in their common binding site,wherein the structure of the bound lincomycin is derived from SA50Slinc(presented in pink) and structure of the bound clindamycin is derivedindependently from PDB ID: 1JZX disclosing D. radiodurans-lincomycincomplex (D50S-CLY, presented in grey), PDB ID: 1YJN disclosing H.marismortui-lincomycin complex (H50S-CLY, presented in sky-blue), andPDB ID: 3OFZ disclosing E. coli-lincomycin complex (E70-CLY, presentedin green);

FIGS. 23A-23B present a graphical illustration of the binding pocket oflincomycin in SA50S, wherein FIG. 23A shows lincomycin (presented inpink) interacts with the PTC A-site by numerous hydrogen bonds (dashedline) with the 23S rRNA (presented in grey), and FIG. 23B is a 90degrees horizontal rotated view of FIG. 23A; and

FIG. 24 present a graphical illustration of a structural superpositionof the PTC in native SA50S (presented in teal) and in SA50S-lincomycincomplex (presented in pink), showing a difference in the position ofnucleotide A2062 in the SA50Slinc towards the spermidine (SPD) comparedto the native structure.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to astructure-based drug design and, more particularly, but not exclusively,to methods for designing species-specific antimicrobial agents based oncrystal structures of pathogenic ribosomal subunits.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

Despite the high sequence similarity of eubacterial rRNA, speciesspecificity has been observed under clinically relevant conditions ingenuine pathogen, particularly in the modes of acquiring antibioticresistance. While conceiving the present invention, it has beenrecognized by the present inventors that designing target specificdrugs, and in particular drugs that target exogenous pathogenicmicroorganism in a host which is symbiotic with other non-pathogenicmicroorganism, would require identifying and elucidating notabledifferences of strategic vulnerabilities between pathogenic andnon-pathogenic microorganisms at the molecular level. Considering thateven small structural differences can affect drug binding modes, it hasbeen recognized that designing such highly specific antibacterial agentswhich act on the protein synthesis mechanism of the pathogenic bacteriumcan be afforded by acquiring experimental crystal structures of at leastsome subunits of the pathogen's ribosome.

Bacteria are grouped into two categories, Gram positive bacteria andGram negative bacteria, based on their cell wall structure and variouscharacteristics stemming therefrom. Among the various differences, thosethat involve interaction with a host organism and those that involveinteraction with antibiotics bear a particular interest frompharmacologic and therapeutic aspects.

Generally, Gram positive bacteria exhibit a smooth 20-30 nm thick cellwall and no outer membrane; while Gram negative bacteria exhibit a wavy8-12 nm thin cell wall and an outer membrane. In general terms of humanhost interactions, Gram positive bacteria is more aerobic and foundtypically on the skin and in the respiratory tract, including mouth,throat and lungs, while Gram negative bacteria is more anaerobic and istypically found in the GI tract.

In terms of virulence and pathogenicity, both types of bacteria may beinvolved in infectious conditions and diseases, with some distinguishingcharacteristics which are not related to the infected site, organ orsub-system. For example, Gram positive bacteria produce primarilyexotoxins that the bacterial cell secretes during its life cycle, whileGram negative bacteria produce primarily endotoxins that affect the hostupon bacterial cell breakdown.

From the pharmaceutical point of view, the above differences areexpressed in different sensitivity of each of the bacterial types todifferent antibiotics under different conditions, including differentmechanisms of antibiotic activity and different mechanism for developingresistance to antibiotics. For example, cephalosporins of the “firstgeneration”, such as cefadroxil, cefazolin, cefalotin, cefalothin andcefalexin, glycopeptides such as teicoplanin, vancomycin, telavancin,dalbavancin and oritavancin, and lipopeptides such as daptomycin, aremore potent against Gram positive bacteria including MRSA; whilecephalosporins of the “second generation” such as cefaclor, cefamandole,cefoxitin, cefprozil and cefuroxime, cephalosporins of the “thirdgeneration” such as cefixime cefdinir, cefditoren, cefoperazone,cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime andceftriaxone, aminoglycosides such as amikacin, gentamicin, kanamycin,neomycin, netilmicin, tobramycin, paromomycin, streptomycin andspectinomycin, polypeptides such as bacitracin, colistin and polymyxinB, and monobactams such as aztreonam are more potent towards Grampositive bacteria.

Out of the pathogenic Gram positive bacteria family, the Gram positivecocci constitute a sub-family of bacteria which is typically pathogenicin humans. It is estimated that members of this family are the cause ofat least a third of all the bacterial infections observed in humans. Asub-family of Gram positive cocci includes Staphylococci bacteria, whichare among the most common bacteria causing human disease; out of whichStaphylococcus aureus (SA) is one of the most common pathogenicbacterium. Pathogenic staphylococci are ubiquitous, and are carried,usually transiently, in the anterior nares of about 30% of healthyadults and on the skin of about 20%. Rates are higher in hospitalpatients and personnel. Staphylococci species such as S. epidermidis areincreasingly associated with hospital-acquired infections; S.saprophyticus causes urinary infections. S. lugdunensis causes invasivedisease with virulence similar to that of S. aureus.

Most staphylococcal diseases are caused by direct tissue invasion, andinclude skin infections, pneumonia, endocarditis, osteomyelitis andseptic arthritis. Staphylococcus aureus is considered as one of the mostpathogenic bacteria in humans, causing skin infections, pneumonia,endocarditis and osteomyelitis. Infections by this pathogen commonlylead to abscess formation. Some strains elaborate toxins that causegastroenteritis, scalded skin syndrome, and toxic shock syndrome.Diagnosis is by Gram stain and culture. Treatment is usually withpenicillinase-resistant β-lactams, but because antibiotic resistance iscommon, vancomycin or other newer antibiotics may be required. Somestrains are partially or totally resistant to all but the newestantibiotics, which include linezolid, quinupristin/dalfopristin,daptomycin, telavancin, dalbavancin, tigecycline, and ceftaroline.

The present inventors have uncovered the conditions required to produce,isolate and crystallize the large ribosomal subunit (50S) from alife-threatening pathogenic bacterium, Staphylococcus aureus (SA), andthereby have successfully crystallized this bacterial ribosomal unit.The large ribosomal subunit (50S) of Staphylococcus aureus (SA) is thefirst bacterial large ribosomal subunit of a real pathogenic bacteriumwhich has been crystallized to date.

The present inventors have further determined the crystal structure ofthe large ribosomal subunit (50S) of Staphylococcus aureus (SA), hereinthroughout referred to interchangeably as the SA50S subunit, and furtherdetermined the crystal structures of complexes thereof with severalantibacterial agents. The obtained crystal structures were comparedamongst themselves and with structures of ribosomal particles from otherspecies, and the insights gained from these comparative structuralanalyses have been used to provide unique knowledge-based tools forunderstanding species specificity, for improving the clinicalperformance of known antibiotics and for designing novel antibioticswhich would interact with known and newly-identified peripheral bindingsites on the target's ribosome. Furthermore, by studying the intricateinteraction between an antibacterial agent and its molecular target,gaining insights on the molecular structural factors that governimmergence of resistance in a pathogen was enabled.

The SA50S subunit is composed of 2 rRNA chains and 26 rProteins chains.Table 1 below provides their types, names, chain ID and SEQ ID NOs. Theactual sequences of these molecules are listed in the Sequence Listingthat accompanies this application.

TABLE 1 Type Name Chain ID SEQ ID NO.: rRNA 23S X 1 rRNA 5S Y 2 rProteinL2 A 3 rProtein L3 B 4 rProtein L4 C 5 rProtein L5 D 6 rProtein L6 E 7rProtein L13 G 8 rProtein L14 H 9 rProtein L15 I 10 rProtein L16 J 11rProtein L17 K 12 rProtein L18 L 13 rProtein L19 M 14 rProtein L20 N 15rProtein L21 O 16 rProtein L22 P 17 rProtein L23 Q 18 rProtein L24 R 19rProtein L25 S 20 rProtein L27 T 21 rProtein L28 U 22 rProtein L29 V 23rProtein L30 W 24 rProtein L32 Z 25 rProtein L34 2 26 rProtein L35 3 27rProtein L36 4 28

Provided herein is the first crystal structure of a ribosomal particlefrom a genuine pathogen, alongside the crystal structures of itscomplexes with linezolid, telithromycin and lincomycin, and twoexemplary pleuromutilin, BC-3205 and lefamulin.

As used herein, the terms “antibiotic(s)”, “AB”, “antibiotic drug(s)”and “antibacterial agent(s)” are used interchangeably to refer to anaturally occurring, synthetic or semi-synthetic substance which isdirected or effective against bacteria.

Further provided herein are some of the specific traits and principleswhich govern the selectivity and resistance of this genuine pathogen,elucidated by analyzing these structures and comparing them with theknown structures of the corresponding non-pathogenic eubacteria andtheir complexes with linezolid, telithromycin and pleuromutilins.Further provided herein are comparisons between specific ribosomalnucleotides and proteins that belong to the antibiotic binding pocketsin the pathogen's ribosomal subunit and its eukaryotic equivalent. Thestudies presented herein have been utilized in the structural definitionof components within the ribosome that can be selectivity utilized forthe design of novel and/or improved antibacterial agents to target theSA's ribosome.

While further reducing the present invention to practice, the inventorshave used sequence and structure alignments of S. aureus large ribosomalsubunit rRNA with its counterparts from other eubacteria, whichindicated high degree of conservation. Nonetheless, structuralcharacterization of this ribosomal particle and of its complexes withtwo clinically used antibiotics and one potential new antibiotic,revealed significant differences which have been contemplated asknowledge for the design of novel and improved antibacterial agents.Analysis of the structures of the unbound and the drug-bound ribosomesversus ribosomes from non-pathogenic eubacteria, revealed specificstructural motifs that may indicate possible features involved inspecies-specific acquirement of resistance and drug binding. These newlyobtained insights provide unique structural tools for the definition ofthe structural features acquiring species specificity, including thedistinction between pathogenic bacteria and the useful bacterial specieswithin the human body.

The present inventors have also identified structural motifs, within theparticle's core and on its periphery, which do not belong to the knownantibiotic binding sites, but may be candidates for the design ofselective drugs against SA. A non-limiting example is the void in thevicinity of erythromycin binding site (see, FIGS. 11A-B) that may beexploited for improving existing macrolide antibacterial agents andnovel binding sites that may be used for the design of new antibacterialagents, thus enriching the pool of potential antibacterial agents withminimized and/or controlled resistance to pathogenic bacteria. In thisrespect, the superiority of the new lead compound, BC-3205, over knownpleuromutilins, that is indicated by its strong binding to SA ribosomeand low MICs, was clearly corroborated by the structural elements of itsbinding mode.

A Composition-of-Matter:

A ribosome, as this term is used herein and in the field of biology andother life sciences, is a complex sub-cellular entity which is the mainpart of natural protein biosynthesis. The term “ribosomal subunit”, asused herein, describes one of the two subunits of a ribosome—the smallsubunit which generally binds the mRNA, and the large subunit whichgenerally binds the tRNA. Each of the small and large ribosomal subunitsis made from a plurality of RNA molecules, referred to as rRNA, andproteins, referred to as rProteins. The Svedberg unit is typically usedto describe the various ribosomal subunits, whereas the unit is used todenote the rate of sedimentation of the subunit during centrifugationrather than its size. For example, prokaryote cells have 70S ribosomes,each consisting of a small (30S) and a large (50S) subunit, andeukaryotes have 80S ribosomes, each consisting of a small (40S) andlarge (60S) subunit.

According to an aspect of some embodiments of the present invention,there is provided a composition-of-matter which comprises a crystallizedform of at least one ribosomal subunit of a pathogenic bacterium.

According to some embodiments of this aspect of the present invention,the composition-of-matter provided herein includes a crystallized formof a large ribosomal subunit of a pathogenic bacterium which is otherthan Escherichia coli.

According to some embodiments of this aspect of the present invention,the pathogenic bacterium is a pathogenic Gram positive bacterium, and/ora pathogenic bacterium exhibiting a degree of 23S rRNA sequence identityof at least 80% compared to the 23S rRNA of Staphylococcus aureus.

As used herein, the term “pathogen”, including all its inflections, suchas “pathogenic”, describes an infectious microorganism, such as abacterium, a fungus or protozoan, which causes disease in its host.According to some embodiments, the pathogen is an infectious bacterium,and more specifically a bacterium that forms an adverse parasiticassociation with one or more other organisms, referred to herein as a“host”. According to some embodiments, the term “pathogenic bacterium”refers to a member of the domain of bacteria, which causes a disease ordisorder in a mammalian host, such as a human.

In some embodiments of the present invention, the pathogenic bacteriumbelongs to the Kingdom of Eubacteria; in some of the embodiments, thepathogenic bacterium belongs to the Phylum of Firmicutes; in some of theembodiments, the pathogenic bacterium belongs to the Class of Bacilli;in some of the embodiments, the pathogenic bacterium belongs to theOrder of Bacillales; in some of the embodiments, the pathogenicbacterium belongs to the Family of Staphylococcaceae; in some of theembodiments, the pathogenic bacterium belongs to the Genus ofStaphylococcus; and in some of the embodiments, the pathogenic bacteriumbelongs to the Species of Staphylococcus aureus (S. aureus, referred toherein as “SA”).

According to some embodiments, the composition-of-matter describedherein includes a crystallized form of a 50S subunit of a pathogenicGram positive bacterium.

According to some of any of the embodiments of the present invention,the pathogenic bacterium is a Gram positive bacterium. Exemplary ofpathogenic Gram positive bacteria genii include Staphylococcus,Streptococcus, Clostridium, Corynebacterium, Enterococcus and Listeria.

In some of any of the embodiments of the present invention, the Grampositive bacterium is a Gram positive cocci bacterium; in some of theembodiments, the Gram positive cocci bacterium is a Staphylococcusbacterium; and in some of the embodiments, Staphylococcus bacterium isStaphylococcus aureus. In some of the embodiments of the presentinvention, the composition-of-matter provided herein comprises the largeribosomal subunit of Staphylococcus aureus (referred to herein as“SA50S”) in a crystallized form, or a crystal of SA50S.

As known in the art, antibacterial agents, also referred to herein andin the art as antibiotics, and in some cases also as antimicrobialagents, are used to treat hosts which have been infected with apathogenic bacterium. Hence, it is noted herein that the pathogenicityof a pathogenic bacterium may also be associated with the capacity todevelop a resistance to one or more antibacterial agents, namely that apopulation of pathogenic bacteria in a host, which is treated with anantibacterial agent, may survive the treatment by either acquiring theability to survive an exposure to the antibacterial agent in the host,or elsewhere prior to the infection event. As known in the art,antibiotic resistance may develop in pathogenic bacteria by mutations inthe bacterial genetic code, which are expressed, for example, in therRNA and ribosomal proteins thereof.

According to some embodiments of the present invention, the ribosomalsubunit forming a part of the composition-of-matter provided herein isof a pathogenic bacterium which is capable of developing a resistance toan antibacterial agent (also referred to herein as “antibiotic resistantpathogenic bacteria”).

In some of any of the embodiments of the present invention, theantibiotic resistant pathogenic bacterium is an antibiotic resistantpathogenic Gram positive bacterium.

In some of any of the embodiments of the present invention, theantibiotic resistant pathogenic bacterium is an antibiotic resistantpathogenic bacterium exhibiting a degree of 23S rRNA sequence identityof at least 80% compared to rRNA of Staphylococcus aureus.

In some of any of the embodiments of the present invention, theantibiotic resistant pathogenic bacterium is an antibiotic resistantpathogenic bacterium exhibiting a degree of 23S rRNA sequence identityof less than 99.9% compared to rRNA of Escherichia coli.

Non-limiting examples of antibiotic resistant pathogenic Gram positivebacteria include Streptococcus, Staphylococcus, Enterococcus,Clostridium, Mycobacterium, Corynebacterium, Coccobacillus and Bacillus.

According to some embodiments of the present invention, the pathogenicGram positive bacterium which is capable of developing a resistance toan antibacterial agent is Staphylococcus aureus, and more specificallythe pathogenic bacterium is Staphylococcus aureus such as, but notlimited to, a methicillin-resistant Staphylococcus aureus (MRSA), anoxacillin-resistant Staphylococcus aureus (ORSA), a vancomycin-resistantStaphylococcus aureus (VRSA) and a vancomycin intermediateStaphylococcus aureus (VISA).

According to some embodiments, the composition-of-matter provided hereincomprises a crystallized form of a 50S subunit of an antibioticresistant pathogenic bacterium exhibiting degree of 23S rRNA sequenceidentity of at least 80% compared to the 23S rRNA of an antibioticresistant Staphylococcus aureus.

Without limitation, it is noted that closely related species, in termsof bacterial phylogeny, are expected to exhibit high degree of ribosomestructure similarity, while phylogenetic relations of bacteria istypically assessed by the degree of rRNA sequence identity. In otherwords, ribosomal structure similarity is expected to be high within agenus and within evolutionary closely related bacterial genii. Withoutbeing bound by any particular theory, it is assumed that the higher theoverall rRNA sequence identity—the higher is the structural similarly ofthe compared large ribosomal subunits.

The 23S rRNA is about 3000 nucleotide long component (chain) forming apart of the large ribosomal subunit (50S) in bacteria. The ribosomalpeptidyl transferase activity resides in domain V of this rRNA, and thisdomain is the most common binding site for antibiotics that inhibittranslation (i.e., large subunit ligands). For example, a well-knownmember of this antibiotic class, chloramphenicol, acts by inhibitingpeptide bond formation, with recent 3D-structural studies showing twodifferent binding sites depending on the species of ribosome. Linezolidand quinupristin-dalfopristin also bind to the 23S rRNA, andcross-resistance has been demonstrated between these antibiotics.

Some phylogenetic analysis conventions in the field are based oncomparing bacterial rRNA sequence of one or more ribosomal subunits,such as the 23S ribosomal RNA which forms a part of the large 50Ssubunit, another long and conserved ribosomal RNA chain, or the entirebacterial rRNA. Table 2 below presents some comparative data pertainingto some non-limiting examples of bacterial species, including the degreeof rRNA identity with respect to that of Staphylococcus aureus 23S rRNA,denotation of X-ray structure availability in terms of the structure'sresolution, the availability of complex structures with ribosomalligands, Pathogenicity in mammals and the family affiliation.

TABLE 2 23S rRNA Exemplary % Availability Species identity Resolution ofcomplex (23S accession w/r/t S. of available structure(s) PathogenicityGram number) aureus structure with ligand in mammals staining/FamilyStaphylococcus 100  3.5 Å YES Pathogenic + aureus (NC_007795)Escherichia 76 3.46 Å YES Partly − coli pathogenic (J01695) Thermus 81 3.5 Å YES Non- − thermophilus pathogenic (AP008226) Deinococcus 74 2.91Å YES Non- evolutionary radiodurans pathogenic Gram negative (AE000513but stains as AE001864- Gram positive AE002092) Streptococcus 85 — —Pathogenic + pneumoniae strain R6 (AE007317 AE008385- AE008568) Bacillus90 — — Pathogenic + subtilis strain AG1839 (CP008698) Clostridium 81 — —Pathogenic + difficile (AM180355) Salmonella 75 — — Pathogenic − bongoristrain NCTC 12419 (FR877557.1)

The term “degree of 23S rRNA sequence identity”, as used herein, refersto the degree of identity of the 23S rRNA sequence between that ofStaphylococcus aureus and that of various pathogenic bacteriacontemplated within the scope of the present invention. The use of thephylogenetic relations based on the 23S rRNA also means that all specieswhich can be compared phylogenetically based on the presence of a 23Sribosomal subunit, are contemplated as part of the scope of theinvention according to the definition of the term “degree of 23S rRNAsequence identity”.

Unless stated otherwise, the term “degree of 23S rRNA sequence identity”refers to the difference in 23S rRNA sequence identity between a 23SrRNA sequence of a species of interest and the corresponding 23S rRNAsequence of Staphylococcus aureus.

In one particular instance the term “degree of 23S rRNA sequenceidentity” is used to signify that a ribosomal subunit is not of aparticular species, such as for example Escherichia coli, by statingthat the degree of 23S rRNA sequence identity of a given species is lessthan 99.9% compared to the corresponding 23S rRNA sequence of, forexample, Escherichia coli.

According to some embodiments of the present invention, the degree of23S rRNA sequence identity of various pathogenic bacteria contemplatedwithin the scope of the present invention is higher than 77%, higherthan 78%, higher than 79%, higher than 80%, higher than 81%, higher than82%, higher than 83%, higher than 84%, higher than 85%, higher than 86%,higher than 87%, higher than 88%, higher than 89%, higher than 90%,higher than 91%, higher than 92%, higher than 93%, higher than 94%,higher than 95%, higher than 96%, higher than 97%, higher than 98%, orhigher than 99%.

According to some embodiments of the present invention, any bacterialspecies which is considered “different than Staphylococcus aureus”, or“other than Staphylococcus aureus”, is identified by degree of 23S rRNAsequence identity which is less than 99.9%, less than 98%, less than97%, less than 96%, less than 95%, less than 94%, less than 93%, lessthan 92%, less than 91%, less than 90%, less than 89%, less than 88%,less than 87%, less than 86%, less than 85%, less than 84%, less than83%, less than 82%, less than 81%, or less than 80%, compared to the 23SrRNA sequence of SA. It is noted herein that the 3D structure of aribosome or any subunit thereof may still be highly similar in twodifferent species, and according to the non-limiting theory ofsequence-structure-activity relationship, a prediction of their degreeof structural similarity can be correlated to their degree of 23S rRNAsequence identity.

According to some embodiments of the present invention, the degree of23S rRNA sequence identity of various pathogenic bacteria contemplatedwithin the scope of the present invention is less than 99.9%, less than99%, less than 98%, less than 97%, less than 96%, less than 95%, lessthan 94%, less than 93%, less than 92%, less than 91%, less than 90%,less than 89%, less than 88%, less than 87%, less than 86%, less than85%, less than 84%, less than 83%, less than 82%, less than 81%, or lessthan 80%, compared to the 23S rRNA sequence of Escherichia coli.

As can be seen in Table 2, the degree of 23S rRNA sequence identity of,for example, Escherichia coli compared to Staphylococcus aureus, is 76%,namely lower than 77%.

Similarly, without limitation, it is noted that closely related species,in terms of bacterial phylogeny/genealogy, are expected to exhibit highdegree of ribosomal protein sequence similarity/homology. In otherwords, ribosomal structure similarity is expected to be high within agenus and within evolutionary closely related bacterial genii. Withoutbeing bound by any particular theory, it is assumed that the higher theoverall ribosomal protein sequence similarity/homology—the higher is thestructural similarly/homology of the compared large ribosomal subunits.

According to some embodiments of the present invention, the degree oflarge subunit ribosomal protein sequence similarity/homology of variouspathogenic bacteria contemplated within the scope of the presentinvention, for any one of the large subunit ribosomal proteins is higherthan 70%, higher than 71%, higher than 72%, higher than 73%, higher than74%, higher than 75%, higher than 76%, higher than 77%, higher than 78%,79%, higher than 80%, higher than 81%, higher than 82%, higher than 83%,higher than 84%, higher than 85%, higher than 86%, higher than 87%,higher than 88%, higher than 89%, higher than 90%, higher than 91%,higher than 92%, higher than 93%, higher than 94%, higher than 95%,higher than 96%, higher than 97%, higher than 98%, or higher than 99%.

According to some embodiments, the composition-of-matter provided hereinincludes a crystallized form of a 50S subunit of Staphylococcus aureus(abbreviated as “SA50S”).

For the sake of simplicity and brevity, the description below refers tothe exemplary large ribosomal subunit of the pathogenic bacteriumStaphylococcus aureus (SA50S), however, it is to be understood thatlarge ribosomal subunits of other pathogenic bacteria, as definedherein, are also contemplated in some of any of the embodiments of thepresent invention, and that the use of the term “SA50S” herein should beregarded as an exemplary representative of a large ribosomal subunit ofany pathogenic bacterium in any one of the embodiments and anycombination thereof.

A composition-of-matter, according to any one of the embodimentsdescribed herein, comprises a three-dimensional physical crystal havinga crystal lattice, and characterized by a crystallographic unit celldimensions (cell constants) and symmetry, and an asymmetric unit. Theunit cell is typically characterized by a crystallographic space group,as this term is known in the art. As these terms are known in the art,applying the symmetry operations on the asymmetric unit (following theoperations of the space group) and applying translations of the unitcell along its edges by its unit cell dimensions, would reconstruct themacroscopic structure of the crystal lattice. In the context of thepresent embodiments, the crystal lattice comprises at least a pluralityof ribosomal subunits; hence the composition-of-matter, according tosome embodiments of the present invention, comprises at least a crystalof a ribosomal subunit; and in embodiments of the present invention, thecomposition-of-matter comprises at least a crystal of the largeribosomal subunit of a pathogenic bacterium, as described herein in anyone of the respective embodiments of the present invention.

According to some of any of the embodiments described herein, thecomposition-of-matter may further comprise a medium or a solution whichis referred to as “mother liquor” and includes a plurality of unboundmolecular entities, such as, without limitation, solvent molecules,ions, solutes, buffers, protein solubility modifiers, freezing pointmodifiers and other additives.

According to some embodiments of the present invention, the crystal inthe composition-of-matter may further include other molecular entitieswhich are arranged in the same lattice as the ribosomal subunit, or bepresent in the media channels and passages between the crystallizedribosomal subunits and not necessarily arranged in a lattice, and whichcan be in chemical or physical association with the ribosomal subunit,or not.

When a ribosomal-bound molecular entity is exogenous to the ribosome,namely, it is not an rRNA strand and not a ribosomal protein chain, nota covalently-bound moiety resulting from port-translational cellularmodifications and not an integral part of the ribosomal subunit, it issaid that the ribosomal subunit and the molecular entity form a complex,and typically the molecular entity is referred to as “a ligand of theribosomal subunit”, and referred to herein as a “ligand” in short. Aribosomal ligand can form a complex with a ribosomal subunit and isexpected to affect (i.e., enhance or inhibit) the activity of theribosome.

Thus, in some of any of the embodiments of the present invention, theribosomal subunit of the composition-of-matter may be associated withone or more ligands via chemical and/or physical interactions. By“associated with” it is meant that the ligand is in chemical or physicalassociation with the ribosomal subunit in the composition-of-matter or aportion thereof; whereas “associated with” is meant to encompass termssuch as “bound to”, “complexed with” and the likes. When associated viachemical interactions, the association may be effected, for example, byone or more covalent bonds and/or by one or more non-covalentinteractions. Examples of non-covalent interactions include hydrogenbonds, electrostatic interactions, donor-acceptor interactions, Van derWaals interactions, metal-coordination interactions andhydrophobic/aromatic interactions. These interactions lead to thechemical association of the ligand to the ribosomal subunit in thecomposition-of-matter. When associated via physical interactions, theassociation may be effected, for example, via absorption, entrapment,and the like.

In some of any of the embodiments of the present invention, anon-covalent or physical association of a ligand molecule with theribosomal subunit is characterized by a dissociation constant of lessthan 10⁻⁵ M. In some of the embodiments, the dissociation constant isless than 10⁻⁶ M. In some of the embodiments, the dissociation constantis less than 10⁻⁷ M. In some of the embodiments, the dissociationconstant is less than 10⁻⁸ M. In some of the embodiments, thedissociation constant is less than 10⁻⁹ M. In some of the embodiments,the dissociation constant is less than 10⁻¹⁰ M.

In some of any of the embodiments, a ribosomal subunit is associatedwith a ligand molecule via hydrophobic interactions, for example, awater-insoluble ligand molecule adheres to a hydrophobic region, e.g. ahydrophobic core or hydrophobic moieties, of a ribosomal subunit.

In some of the embodiments of the present invention, a ligand iscovalently bound to the ribosomal subunit.

It is further assumed, without limitation, that the ligand, e.g. theantibacterial agent, binds to the ribosomal subunit in its crystallizedform substantially in the same binding site on the ribosomal subunit andsubstantially by the same mode of binding (orientation, affinity and thelikes) as in its solubilized (un-crystallized) form and substantiallythe same as it would bind to the ribosomal subunit in the living cell.Exemplary ligands include small molecules (less than 1 kD molecularweight), non-ribosomal peptides and polypeptides, carbohydrate,non-ribosomal nucleic acids and combination thereof. Ligands can play arole of co-factors, active-site inhibitors, activators and generalribosomal activity modulators.

In some of any of the respective embodiments of the present inventiondescribed herein, a ligand is an exogenous molecular entity that iscapable of penetrating into the bacterial cell and forming a complexwith the ribosome while the cell is still functional (live), therebyaffecting the cell functions, primarily the cellular protein synthesisactivity. In the context of some embodiments of the present invention,when the ligand inhibits the ribosomal activity, such as proteinsynthesis, it is referred to as an antibiotic or antibacterial agent.

The aforementioned complex can form before the crystal is formed, or beintroduced into a pre-formed crystal of the ribosomal subunit, and formthe complex while substantially preserving the crystal lattice and otherproperties of the pre-formed crystal such as unit cell dimensions andoverall molecular arrangement and fold. It is noted that any molecularentity which is bound to the crystallized ribosomal subunit, forms apart of a crystallized complex, which is to say that it is found in thecrystal lattice of the ribosomal subunit. It is noted that the unit celldimensions may vary from crystal to crystal stemming from the same batchof crystals depending on environmental factors and the mother liquorcondition and composition. Such variations in cell dimensions may be inthe order of up to 1-4% in cell edge length. For example, when a unitcell dimension is about 300 Å it can vary from sample to sample (crystalto crystal) by about ±10 Å.

In some of any of the respective embodiments of the present inventiondescribed herein, the identification of a ligand with respect to itstarget of binding is made based on size differences, namely the largerentity is regarded as the target, and the smaller entity is the ligand.It is further noted that the term “ligand”, as used herein, typicallyrefers to molecular entities that bind in relatively small numbers(number of identical ligands per one major binding target molecularentity) to a specific site on the target and affect its biologicactivity.

According to some of any of the embodiments of the present invention,the molar ratio of the ribosomal subunit in the lattice of thecomposition-of-matter to the bound ligand ranges from 1:1 to 1:2, orfrom 1:1 to 1:4, or from 1:1 to 1:6. According to some embodiments,solvent (e.g., water, ethanol, etc.) molecules, salt ions and othersolutes which may bind to many sites in and/or on the ribosomal subunit,are not regarded as ligands since the ratio of ribosomal subunit to suchmolecular entities is typically larger than 1:6.

As demonstrated in the Examples section that follows, the presentinventors have successfully prepared a sample of a substantiallypurified large ribosomal subunit obtained from the pathogenic bacteriumStaphylococcus aureus, and further crystallized this large ribosomalsubunit following a crystallization procedure, which is described indetails hereinbelow.

According to some of any of the embodiments of the present invention,the crystallized large ribosomal subunit obtained from Staphylococcusaureus is characterized by the crystal space group P6522, and furthercharacterized by a unit cell dimensions of a=279.756±10 Å, b=279.756±10Å, c=872.725±10 Å, α=90, β=90 and γ=120.

It is noted that large ribosomal subunits obtained from other pathogenicbacteria, and also large ribosomal subunit obtained from Staphylococcusaureus and subjected to different crystallization procedures, may formcrystals having a different crystal lattice, difference space groupsymmetry and different unit cell dimensions, and therefore theaforementioned crystal space group and unit cell dimensions should notbe seen as limiting the scope of the invention, but rather as anembodiment thereof.

As discussed hereinabove, the crystallized entity in thecomposition-of-matter provided herein may be the ribosomal subunit perse or a complex of the ribosomal subunit and a ligand thereof.

According to some of any of the embodiments of the present invention,the composition-of-matter comprises a crystallized complex of aribosomal subunit and an antibacterial agent. Without being bound to anyparticular theory, it is assumed that the mechanism by which theantibiotic agent exerts its antibiotic activity is by binding to theribosomal subunit, thereby interfering with the biosynthesis ofbacterial proteins.

According to some of any of the embodiments of the present invention,the composition-of-matter comprises a ligand which belongs to a familyof ribosomal-active antibacterial agents, such as, but not limited to,the oxazolidinones family, the pleuromutilins family, and the macrolidefamily.

According to some embodiments of the present invention, exemplaryantibacterial agents which form a crystallized complex in thecomposition-of-matter provided herein, include, without limitation,linezolid (an oxazolidinone), BC-3205 (a pleuromutilin) andtelithromycin (a macrolide or a ketolide).

As presented in the Examples section that follows, the crystallizedcomplex of the large ribosomal subunit of Staphylococcus aureus withlinezolid is characterized by a crystal space group of P6522 and a unitcell dimensions of a=279.922±10 Å, b=279.922±10 Å, c=870.586±10 Å, α=90,β=90 and γ=120; the crystallized complex of the large ribosomal subunitof Staphylococcus aureus with BC-3205 is characterized by a crystalspace group of P6522 and a unit cell dimensions of a=280.918±10 Å,b=280.918±10 Å, c=875.585±10 Å, α=90, β=90 and γ=120; and thecrystallized complex of the large ribosomal subunit of Staphylococcusaureus with telithromycin is characterized by a crystal space group ofP6522 and a unit cell dimensions of a=282.66±10 Å, b=282.66±10 Å,c=877.075±10 Å, a=90, β=90 and γ=120.

According to some of any of the embodiments of the present invention,any of the crystallized large ribosomal subunit of Staphylococcus aureusprovided herein, either native (no ligand associated therewith) orcomplexed (associated) with an antibacterial agent, is characterized bya crystal lattice order which is sufficient to diffract collimated andfocused electromagnetic radiation effectively so as to allow thisdiffracted radiation to be detected, measured and analyzed. Thus, any ofthe crystallized large ribosomal subunit of Staphylococcus aureusprovided herein effectively diffracts X-rays for determination of atomiccoordinates to a resolution of at least 10 Å, 8 Å, 6 Å, 5 Å, 4.5 Å, 4 Å,3.5 Å or at least 3 Å.

Crystallization of a Large Ribosomal Subunit of a Pathogenic Bacterium:

According to an aspect of some embodiments of the present invention, thecomposition-of-matter described herein is afforded by crystallization ofa purified sample of a ribosomal subunit, such as the large ribosomalsubunit, of a pathogenic bacterium, and/or a pathogenic Gram positivebacterium, and/or a pathogenic bacterium exhibiting degree of 23S rRNAsequence identity of at least 80% compared to the 23S rRNA ofStaphylococcus aureus. In some of any of the respective embodiments ofthe present invention, the composition-of-matter described herein isafforded by crystallization of a purified sample of the large ribosomalsubunit of Staphylococcus aureus, also referred to herein as the SA50Sribosomal subunit.

According to some embodiments of the present invention, the sample isobtained by harvesting and purifying ribosomes from a lysate of aculture of the pathogenic bacterium under study. Purification andconcentration of the ribosomal subunits is typically effected bycentrifugation and/or chromatographic techniques and/or dialysis, all ofwhich under conditions that preserve the structure of the ribosomalsubunits as assessed, for example, by their ability to exert ribosomalactivity.

The crystallization of the purified and concentrated ribosomal subunitis carried out by any method known in the art. An exemplary proteincrystallization technique known as “hanging drop vapor diffusioncrystallization” involves a downwards facing drop composed of a mixtureof the protein/ribosome sample and a variety of reagents which is placedinside a vapor equilibration enclosure comprising a reservoir ofreagents solution. Another exemplary protein crystallization techniqueis known as “sitting drop vapor diffusion crystallization” and involvesan upwards facing drop composed of a mixture of the protein/ribosomesample and a variety of reagents which is placed inside a vaporequilibration enclosure comprising a reservoir of reagents solution.Another exemplary protein crystallization technique is known as“crystallization under oil” and involves a drop of sample combined withthe crystallization reagent of choice which is pipetted under a layer ofoil (also known as “microbatch crystallization”), whereas the oils canalso be used as a barrier between the reservoir and the drop intraditional hanging or sitting drop crystallization experiments withvapor diffusion rate control. Another exemplary protein crystallizationtechnique is known as “dialysis crystallization”, which is not a vapordiffusion technique, and involves a sample separated from a precipitantsolution by a semi-permeable membrane that allows small molecules suchas ions, additives, buffers and salts to pass but prevent biologicalmacromolecules from crossing the membrane.

According to some embodiments of the present invention, a sample ofpurified and concentrated SA50S ribosomal subunit is subjected tocrystallization by a vapor diffusion technique, or vapor diffusionconditions, in which a solvent is gradually depleted by vaporizationfrom a crystallization solution comprising the purified and concentratedSA50S sample and a precipitant (a substance that causes precipitationwhen added to a solution). During the vapor diffusion process theconcentration of the precipitant in the crystallization solutiongradually increases, thereby gradually lowering the solubility of theSA50S in the crystallization solution, such that the crystallizationsolution becomes saturated with respect to the SA50S. During the vapordiffusion process the SA50S gradually de-solubilizes (comes out ofsolution) to precipitate in an orderly fashion and deposit in athree-dimensional lattice to form a crystal thereof. Thus, according tosome embodiments, the method of obtaining a crystal of SA50S is effectedby subjecting an aqueous solution of SA50S to vapor diffusionconditions.

The phrase “vapor diffusion conditions”, as used herein, refers tochemical, physical, mechanical and/or environmental conditions thatfavor crystal growth. In some of the embodiments of the presentinvention, slow, gradual, uninterrupted and controlled increase of theconcentration of the entity to be crystallized allows the entity to forma lattice rather than amorphous sediment. In some of the embodiments,vapor diffusion conditions are such that the crystallization solutionand the reservoir solution are kept in a relatively small (1-10 ml)container that can be essentially sealed-off the ambient environment.

In some of the embodiments of the present invention, the volume of thecrystallization solution is significantly smaller than the volume of thereservoir solution (so as to, e.g., drive the equilibration process moreefficiently).

In some of the embodiments of the present invention, the volume of thecrystallization solution ranges from about 1 microliter (μl) to about100 microliters (μl), and the volume of the reservoir solution rangesfrom about 0.1 microliter (ml) to about 10 milliliters (ml).

In some of the embodiments of the present invention, the crystallizationsolution is filtered prior to the crystallization process, so as, forexample, to remove microscopic particles and/or minimize the formationof small crystals and/or minimize the formation of amorphous sediments.

In some of the embodiments of the present invention, the vapor diffusionconditions further include stabilized, constant and controlledtemperature and humidity in a vibration-free environment.

In some of the embodiments of the present invention, the vapor diffusionconditions include sealing the crystallization solution in a compartmentthat also includes a reservoir of another solution, referred to hereinas a “reservoir solution”.

According to some embodiments of the present invention, the mechanicalarrangement of the crystallization solution and the reservoir solutiontakes the format of a hanging drop arrangement or a sitting droparrangement. In some of the embodiments, the vapor diffusion conditionstake the format of a hanging drop in which a drop of the crystallizationsolution is applied substantially at the center and bottom of anair-tight lid (typically a transparent and circular cover slip) thatcloses a top opening of a container holding the reservoir solution.According to some embodiments, the volume of the crystallizationsolution (the volume of the hanging drop) is about 2-8 μl and the volumeof the reservoir solution is about 0.8-1.2 ml.

According to some of any of the embodiments of the method described inthis context of the present invention, the aqueous solution of SA50Swhich is subjected to vapor diffusion conditions is referred to hereinas a “crystallization solution”. In some of the embodiments, the SA50Sin the crystallization solution is concentrated to a range of, forexample, from 0.8 mg/ml to 2 mg/ml, or from 1 mg/ml to 1.6 mg/ml,including any subranges and intermediate values between these ranges. Insome of the embodiments, the solution of SA50S is concentrated by meanscommonly used in the art, such as, for example, centrifugation,filtration, dialysis and the likes.

According to some of any of the embodiments of the method described inthis context of the present invention, the crystallization solution mayfurther include one or more additives which stabilize the SA50S particlein terms of viability of its proteins and RNA constituents. In some ofthe embodiments, the additives include buffers, salts, oxidationretardants and the likes, which are used to set or control propertiessuch as ionic strength, pH, chemical composition and the likes. Theseadditives also promote structural stability and viability of variousfunctional groups present in or on the SA50S.

Exemplary crystallization solution additives include, withoutlimitation, non-specific and non-ribosomal antimicrobial agents such assodium azide, reducing agents that preserve sensitive or labile chemicalbonds such as tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT)or 2-mercaptoethanol, which preserves disulfide bonds, ribosomalstabilizers such as spermidine, ionic agents such as MnCl₂, MgCl₂, NH₄Cland KCl, pH buffering agents such as Hepes buffer, alcohols and polyolssuch as ethanol and 2-methyl-2,4-pentanediol.

Other additives that can be used in the crystallization solutionaccording to some embodiments of the present invention include, withoutlimitation, multivalent salts such as barium chloride dihydrate, cadmiumchloride hydrate, calcium chloride dihydrate, chromium(III) chloridehexahydrate, cobalt(II) chloride hexahydrate, copper(II) chloridedihydrate, iron(III) chloride hexahydrate, magnesium chloridehexahydrate, manganese(II) chloride tetrahydrate, nickel(II) chloridehexahydrate, praseodymium(III) acetate hydrate, strontium chloridehexahydrate, yttrium(III) chloride hexahydrate, zinc chloride; saltssuch as ammonium sulfate, cesium chloride, lithium chloride, potassiumchloride, potassium sodium tartrate tetrahydrate, sodium chloride,sodium citrate tribasic dihydrate, sodium fluoride, sodium iodide,sodium malonate and sodium thiocyanate; dissociating agent such asphenol, dimethyl sulfoxide and sodium bromide; a linker such as6-aminohexanoic acid, 1,5-diaminopentane dihydrochloride,1,6-diaminohexane, 1,8-diaminooctane, glycine, glycyl-glycyl-glycine,taurine and betaine hydrochloride; a polyamine such as spermidine,spermine tetrahydrochloride, hexammine cobalt(iii) chloride andsarcosine; a chaotrope such as trimethylamine hydrochloride, guanidinehydrochloride and urea; a co-factor such as β-nicotinamide adeninedinucleotide hydrate and adenosine-5′-triphosphate disodium salthydrate; a Reducing Agent such as TCEP hydrochloride, GSH (L-Glutathionereduced) and GSSG (L-Glutathione oxidized); a polymer such aspolyvinylpyrrolidone K15, dextran sulfate sodium salt, pentaerythritolethoxylate (3/4 EO/OH) and polyethylene glycol (PEG); a carbohydratesuch as D-(+)-glucose monohydrate, sucrose, xylitol, D-sorbitol,myo-Inositol, D-(+)-trehalose dehydrate and D-(+)-galactose; a polyolsuch as thylene glycol and glycerol; and other volatile and non-volatileorganic additives such as benzamidine hydrochloride,n-dodecyl-N,N-dimethylamine-N-oxide, n-octyl-β-D-gluco side,n-dodecyl-β-D-maltoside, trimethylamine N-oxide dihydrate,1,6-hexanediol, (+/−)-2-methyl-2,4-pentanediol, polyethylene glycol 400,jeffamine M-600, 2,5-hexanediol, (±)-1,3-butanediol, polypropyleneglycol P 400, 1,4-dioxane, ethanol, 2-propanol, methanol,1,2-butanediol, tert-butanol, 1,3-propanediol, acetonitrile, formamide,1-propanol, ethyl acetate, acetone, dichloromethane, 1-butanol,2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoro-2-propanol.

The use of additives in the crystallization solution is well known to aperson of ordinary skills in the art of protein crystallization.Alternatively, one can follow guidelines provided in, for example,“Sparse matrix sampling: a screening method for crystallization ofproteins” [Jancarik, J. and Kim, S.-H., J. Appl. Cryst., 1991, 24(4), p.409-411], or in “Improved Success of Sparse Matrix ProteinCrystallization Screening with Heterogeneous Nucleating Agents” [Thakur,A. S. et al, PLoS One, 2007, 31, 2(10)].

Exemplary components that can be added to the crystallization solutionof SA50S include, without limitation, spermidine (e.g., at aconcentration ranging from 2 mM to 10 mM, or from 4 mM to 6 mM or of 5mM), MnCl₂ (e.g., at a concentration ranging, e.g., from 0.1 mM to 1 mM,or from 0.4 mM to 0.6 mM or of 0.5 mM), 20 mM Hepes buffer (e.g., at aconcentration ranging from 5 mM to 150 mM, or of 10 mM, 20 mM, 50 mM, 80mM or 110 mM) set to pH range of from 6.8 to 7.8, MgCl₂ (e.g., at aconcentration ranging from 1 mM to 20 mM, or of 5 mM, 10 mM or 15 mM),NH₄Cl (e.g., at a concentration ranging from 20 mM to 80 mM, or of 30mM, 40 mM, 50 mM or 60 mM) and/or KCl (e.g., at a concentration rangingfrom 5 mM to 30 mM, or of 5 mM, 10 mM, 15 mM or 20 mM).

According to exemplary, non-limiting, embodiments, the crystallizationsolution comprises a concentrated purified preparation of SA50S at aconcentration ranging from 0.9 mg/ml to 1.6 mg/ml,2-methyl-2,4-pentanediol (MPD) at a concentration ranging from 0.1weight percent to 0.2 weight percent, ethanol at a concentration rangingfrom 0.3 weight percent to 0.4 weight percent, spermidine at aconcentration ranging from 4 mM to 6 mM, MnCl₂ at a concentrationranging from 0.4 mM to 0.6 mM, 20 mM Hepes buffer set to pH range of6.8-7.8, 10 mM MgCl₂, 60 mM NH₄Cl and 15 mM KCl.

According to some of any of the embodiments of the method described inthis context of the present invention, the vapor diffusion conditionsinclude a crystallization solution, as described in any one of therespective embodiments, and/or a reservoir solution, as described in anyone of the respective embodiments, having one or more solubilized yetnon-volatile component(s), referred to herein as precipitant(s). In someof any of the respective embodiments, the participant(s) is/are found ata higher concentration in the reservoir solution compared to thecrystallization solution, such that the gradient of concentration of theprecipitant(s) drives a volatile solvent to diffuse via vapor phase fromthe crystallization solution to the reservoir solution. In some of theseembodiments, the concentration of the precipitant in the reservoirsolution is higher by at least 1%, 5%, 10%, 20%, 30%, 50%, 100% or 200%or higher, compared to the concentration of the precipitant in thecrystallization solution.

Exemplary precipitants usable in the context of the present embodimentsinclude, but are not limited to, a salt, an alcohol, a polyol, a glycol,glycerol, a polyglycol, a polyethylene glycol (PEG) and any mixturethereof. According to some embodiments, the precipitant comprisesammonium sulfate, ethanol and 2-methyl-2,4-pentanediol (MPD), andaccording to some embodiments, the precipitant comprises ethanol andMPD.

According to some embodiments, the concentration of MPD in thecrystallization solution ranges from 0.1 weight percent to 0.3 weightpercent, or from 0.15 weight percent to 0.2 weight percent. According tosome embodiments, the concentration of ethanol in the crystallizationsolution ranges from 0.2 weight percent to 0.5 weight percent, or from0.3 weight percent to 0.4 weight percent. In some of any of therespective embodiments, the crystallization solution includes 0.166% MPDand 0.333% EtOH.

According to some of any of the embodiments of the method described inthis context of the present invention, the reservoir solution comprisesfrom 5 weight percent to 15 weight percent MPD, or from 8 to 12 weightpercent MPD, and from 3 weight percent to 7 weight percent ethanol, orfrom 4 weight percent to 6 weight percent ethanol. In some of any of therespective embodiments, the reservoir solution includes 5 weight percentethanol and 10 weight percent MPD.

Both the crystallization and reservoir solutions further include from 10mM to 120 mM Hepes buffer set to pH range of 6.8-7.8, from 5 mM to 15 mMMgCl₂, from 50 mM to 70 mM NH₄Cl and from 10 mM to 20 mM KCl.

According to some of any of the embodiments of the method described inthis context of the present invention, under vapor diffusion conditions,the concentration of the precipitant(s) in the crystallization solutionincreases, thereby driving the ribosomal subunit to graduallyprecipitate and crystallize over a sufficient period of time, referredto herein as a “first time period”.

According to some of any of the embodiments of the method described inthis context of the present invention, the first time period ranges from0.5 day (12 hours) to 60 days, or from 1 day to 10 days, from 2 days to20 days, from 3 days to 30 days, or from 5 days to 50 days. In some ofthe respective embodiments, the first time period ranges from 10 days to20 days.

Once the crystallization solution comes to equilibrium with respect tothe reservoir solution, and crystals are formed therein, thecrystallization solution is referred to as “mother liquor”.

According to some of any of the embodiments of the method described inthis context of the present invention, the reservoir solution includessome or all of the components of the crystallization solution. In someof these embodiments, the reservoir solution differs from thecrystallization solution by one or more of the concentration of aprecipitant, as described herein, and the presence of the entity that iscrystallized (the large ribosomal unit). In some of the respectiveembodiments, the reservoir solution does not contain the entity to becrystallized.

An exemplary crystallization solution which affords SA50S crystalscomprises concentrated purified preparation (SA50S) at a concentrationthat ranges from 0.9 mg/ml to 1.6 mg/ml, from 0.1 weight percent to 0.2weight percent 2-methyl-2,4-pentanediol, from 0.3 weight percent to 0.4weight percent ethanol, from 4 mM to 6 mM spermidine, from 0.4 mM to 0.6mM MnCl₂, 20 mM Hepes buffer set to pH range of 6.8-7.8, 10 mM MgCl₂, 60mM NH₄Cl and 15 mM KCl.

According to some of any of the embodiments described for the method ofthis aspect of the present invention, the method further comprisesextracting a plurality of SA50S crystals at the end of the first timeperiod, and subjecting these crystals to conditions that allow thesecrystals to grow further in size. These crystals, which are extracted inorder to allow them to further grow, are referred to as “seedingcrystals”. It is noted that for structure elucidation using X-raydiffraction data, a large crystal is advantageous as the intensity ofthe diffracted X-ray is proportional to the size of the crystal.

According to some embodiments of the present invention, the seedingcrystals are transferred into a fresh crystallization solution, preparedas described hereinabove. The fresh crystallization solution, referredto herein as a “re-crystallization solution”, can be the same as theinitial crystallization solution or different in terms of its contentsor the concentration of one or more of its components. For example, thecontent of the ribosomal subunit can be higher or lower than itsconcentration in the initial crystallization solution.

According to some embodiments of the present invention, the seedingcrystals are washed before being transferred to the re-crystallizationsolution. In the context of embodiments of the present invention,washing the crystals refers to contacting the seeding crystals with asolution which is similar to the mother liquor or the reservoir solutionbut does not contain the entity to be crystallized (e.g., a ribosomalsubunit). Without being bound to a particular theory, it is assumed thatwashing the seeding crystals in the abovementioned washing solutioninvolves re-solubilizing some of the particles on the faces of theseeding crystals, thereby exposing fresh and ordered parts of thecrystal lattice. The freshly exposed lattice will serve for growth ofthe crystal in an orderly fashion by essentially extending thepre-formed lattice of the seeds.

According to some of any of the embodiments of the method described inthis context of the present invention, the washing solution comprisesfrom 7 weight percent to 8 weight percent MPD, and from 7 weight percentto 8 weight percent ethanol. The washing solution may further includesome of the components present in the crystallization solution, such as,for example, 110 mM Hepes buffer set to pH range of 6.8-7.8, 10 mMMgCl₂, 60 mM NH₄Cl and 15 mM KCl buffer and 0.5 mM MnCl₂.

According to some of any of the embodiments described for the method ofthis aspect of the present invention, the method further comprisessubjecting the re-crystallization solution which includes one or moreseeding crystals, to vapor diffusion conditions for a second timeperiod. In some of the respective embodiments described herein, thesecond time period typically ranges from 1 day to 60 days, or from 1 dayto 10 days, from 2 days to 20 days, from 3 days to 30 days, or from 5days to 50 days, or from 10 days to 50 days, including any subranges andintermediate values therebetween. In some of the respective embodiments,the second time period ranges from 10 days to 40 days or from 2 weeks to4 weeks.

Seeding crystals of SA50S are placed, after the optional washing step,in a re-crystallization solution which is allowed to equilibrate againsta reservoir solution in essentially the same vapor diffusion conditionsdescribed herein.

According to some embodiments, the SA50S crystals, namely the seedingcrystals and/or the seeded and enlarged SA50S crystals (macro crystal),are further transferred to a stabilization solution after the first orsecond time periods in order to maintain the crystals soaked at optimalconditions and ready for exposure to X-ray radiation. Typically, thestabilization solution contains a higher precipitant concentration whichkeeps the SA50S (the crystallized particles) from re-solubilizing.

According to some of any of the embodiments of the method described inthis context of the present invention, the precipitant in thestabilization solution comprises from 10 weight percent to 20 weightpercent MPD and from 10 weight percent to 20 weight percent ethanol. Insome of the respective embodiments, the stabilization solution comprisesfrom 10 weight percent to 25 weight percent MPD, from 10 weight percentto 20 weight percent ethanol, and further includes other components ofthe crystallization solution, such as, for example, 110 mM Hepes bufferset to pH range of 6.8-7.8, 10 mM MgCl₂, 60 mM NH₄Cl and 15 mM KCl.

According to some embodiments of the present invention, the crystal isflash-cooled (flash-frozen) to cryogenic temperatures and the X-raydiffraction data collection is carried out under cryogenic conditions.In such embodiments, the SA50S crystals are typically soaked in astabilization solution that further contains a cryoprotectant.

The term “cryoprotectant”, as used herein, refers to a substance that isadded to an aqueous solution containing a sample, such as a biologicalsample, in order to protect the sample from rapid formation of extendedice crystals therein, otherwise referred to as freezing damage. Hence,the term “cryoprotectant solution” is used herein to refer to astabilization solution which includes a cryoprotectant.

According to some of any of the embodiments of the method described inthis context of the present invention, the cryoprotectant is an alcohol,a polyalcohol, a saccharide (a carbohydrate), a polysaccharide, apolyol, a glycol, a polyglycol, a polyethylene glycol (PEG) and anymixture thereof. Exemplary cryoprotectants include, without limitation,DMSO, ethylene glycol, glycerol, MPD, propylene glycol and sucrose. Insome of the respective embodiments, the cryoprotectant includes ethanoland MPD.

According to some of any of the embodiments of the method described inthis context of the present invention, prior to the cryogenicflash-freezing, the crystals of the ribosomal subunit, e.g., SA50S, aresoaked in a cryoprotectant solution. According to some embodiments, theprecipitants are similar to the cryoprotectants, and in some of therespective embodiments the stabilization solution can serve as acryoprotectant solution. In some of the respective embodiments, theconcentration of the precipitants/cryoprotectants is adjusted so as toafford cryogenic protection to the ribosomal subunit crystals, typicallyby using higher concentration of the precipitants in the cryoprotectantsolution.

According to some embodiments, the precipitant in the cryoprotectantsolution comprises from 10 weight percent to 25 weight percent MPD andfrom 10 weight percent to 20 weight percent ethanol, and furtherincludes other components of the crystallization solution, such as, forexample, 110 mM Hepes buffer set to pH range of 6.8-7.8, 10 mM MgCl₂, 60mM NH₄Cl, 15 mM KCl and 0.4-0.6 mM MnCl₂.

According to some embodiments of the present invention, the ribosomalsubunit, e.g., SA50S, is further associated with (complexed with) aligand that diffuses into the crystal and is associated essentially withthe crystallized ribosomal subunits. According to some embodiments, theligand is added to the stabilization solution, whereas the stabilizationsolution that comprises a ligand is referred to herein as a “soakingsolution”.

According to some of any of the embodiments of the method described inthis context of the present invention, the macro crystals are soaked inthe soaking solution in order to introduce a ligand to the ribosomalsubunit in the crystals. In some of the respective embodiments, theligand is as defined in any of the respective embodiments describedherein. In some of the respective embodiments, the ligand is anantibacterial agent that binds to a binding site in the crystallizedribosomal subunit. In some of the respective embodiments, theconcentration of the ligand in the soaking solution ranges from 1 μg/mlto 30 μg/ml.

An exemplary crystallization method of the SA50S ribosomal subunit isdescribed in the Examples section that follows. The crystallizationmethod exemplified below has been used to obtain a composition-of-mattercomprising crystals of the large ribosomal subunit of Staphylococcusaureus which were used to obtain X-ray diffraction data to resolution ofat least 4 Å.

The exemplary SA50S crystals in an exemplary composition-of-matter asdescribed herein can be used to obtain X-ray diffraction data accordingto any method, process or X-ray source as known in the art. According tosome embodiments, the data collection is carried out using a collimatedX-ray beam at a high brightness synchrotron station.

A Computer System Comprising Structural Information:

The X-ray diffraction data collected using the herein-providedcomposition-of-matter that comprises a crystallized form of a largeribosomal subunit of a pathogenic bacterium (excluding Escherichiacoli), a pathogenic Gram positive bacterium, or a pathogenic bacteriumexhibiting degree of 23S rRNA sequence identity of at least 80% comparedto the 23S rRNA of Staphylococcus aureus, such as the SA50S subunit, canbe stored, processed and analyzed on a computer system that can furtherbe used to elucidate the three-dimensional structure thereof.

In some of any of the respective embodiments of the present invention,the X-ray diffraction data is subjected to a computational molecularreplacement procedure which assigns a phase to each of the diffractedreflections. In some of the embodiments, the molecular replacementprocedure utilizes an experimentally-obtained or a calculated structureof a 50S subunit of another species, such as, but not limited to, thestructure of the 50S subunit of D. radiodurans (D50S), having PDB ID:2ZJR, which is used as a starting model.

The computer system can further be used, after the molecular replacementprocedure, to provide positioning data indicative of atomic coordinatesof the large ribosomal subunit. Typically, atomic coordinates areobtained by following a procedure wherein the computational atomic modelof the large ribosomal subunit is built computationally into a graphicrepresentation of the electron density map, which is computationallyconstructed from the experimentally recorded intensities and thecalculated phases of the X-ray reflections. Then, an atomic model iscomputationally refined using a computational refinement procedure thatfurther improves the calculated estimation of the phases iteratively,leading to a better defined calculated electron density map.

Thus, the computer system includes positioning data, which define thestructural information of the large ribosomal subunit, either in itsnative form or in its ligand-bound state (a complex with a ligand),being recorded on one or more data-storage device forming a part of thecomputer system. The computer system includes software (computerprograms) that can be used to compare that structural information tostructural information of other ribosomal particles, provide and useprotein and RNA sequence alignments and use the latter to model variousstructures, identify and analyze the molecular surface of variousbinding sites in the large ribosomal subunit, and provide structuraldata pertaining to putative ligands of the large ribosomal subunit.

According to some embodiments of the present invention, the atomiccoordinates of the exemplary native (ligand-free) SA50S subunit aredeposited at the Protein Data Bank under accession number PDB ID: 4WCE.It is noted herein that the phrase “atomic coordinates which aredeposited at the Protein Data Bank under accession number PDB ID: 4WCE”refers to an embodiment of the positioning data pertaining to the native(ligand-free) SA50S ribosomal subunit, which has been determined usingX-ray diffraction data obtained by using a composition-of-matterdisclosed herein. It is further noted that the contents of the entrydeposited at the Protein Data Bank under accession number PDB ID: 4WCEare provided herein, as an electronic document denoted“4WCE_SA50S_Native.pdb.txt”.

Thus, according to some embodiments of an aspect of the presentinvention, there is provided a computer system that includes, withoutlimitation:

(a) at least one data-storage device having stored therein positioningdata indicative of atomic coordinates of a large ribosomal subunit of apathogenic bacterium, wherein the pathogenic bacterium is a pathogenicGram positive bacterium; and/or a pathogenic bacterium exhibiting adegree of 23S rRNA sequence identity of at least 80% compared to rRNA ofStaphylococcus aureus; and/or a pathogenic bacterium exhibiting a degreeof 23S rRNA sequence identity of less than 99.9% compared to rRNA ofEscherichia coli, such as, for example, PDB ID: 4WCE which contains theatomic coordinates of the SA50S ribosomal subunit, which can bedetermined from an electron density map having a resolution of at least4 Å calculated from X-rays diffraction data obtained using at least onecrystal in the composition-of-matter presented herein; and

(b) a processing unit in electrical communication with the data-storagedevice; and

(c) a program for calculating a three-dimensional model representativeof the ribosomal subunit from the set of atomic coordinates.

The computer system may further include a device for providing a visualrepresentation of the three-dimensional model, namely a display(screen). The visual display may present the three-dimensional model ofthe instantly provided SA50S structure as an electron density map, aball-and-stick model, a space-filling (Calotte) model, a secondarystructure (ribbon) model, a molecular surface model, a skeletal modeland the likes and any combination thereof, each of which is aimed atshowing the relative positions of various attributes the atoms making upthe SA50S subunit as captured in the crystal structure. The computersystem is configured to present the entire SA50S or any parts andportions thereof, and further display entire or part of structures ofother molecular entities, including ligands, putative designed ligandsand/or structures of other ribosomal subunits in any combination withthe molecular model of the SA50S subunit.

The computer system may further include various components which formpart of the system and include, without limitation, input/outputcontrollers and devices, memory modules, hard drives, discs and otherform of magnetic and optic data storage controllers and devices, variousports and connectivity controllers and devices, network controllers anddevices and the likes. It is noted that the computer system presentedherein may be wholly or partially operated as could computing system(decentralized computing system), having one or more remote serversnetworked therewith for decentralized data storage and processing, andother forms of online access to decentralized computer services ordecentralized resources.

According to some embodiments of the present invention, the computersystem further includes the atomic coordinates of complexes of the largeribosomal subunit with various ligands bound thereto, such asantibacterial agents. Exemplary ligands include antibacterial agentssuch as linezolid, BC-3205 and telithromycin, as these are deposited atthe Protein Data Bank under accession number selected from the groupconsisting of PDB IDs: 4WFA, 4WFB and 4WF9, respectively. It is notedherein that the phrase “atomic coordinates which are deposited at theProtein Data Bank under accession number PDB IDs: 4WFA, 4WFB and 4WF9”refers to an embodiment of the positioning data pertaining to the SA50Sribosomal subunit in a complex (bound state) with each of the ligandslinezolid, BC-3205 and telithromycin respectively, which have beendetermined using X-ray diffraction data obtained by using acomposition-of-matter disclosed herein.

It is further noted that the contents of each of the entries depositedat the Protein Data Bank under accession number PDB IDs: 4WFA, 4WFB,4WF9, 5HL7 and 5HKV are provided herein, each as an electronic documentdenoted “4WFA_SA50S_lin.pdb.txt”, “4WFB_SA50S_bc3.pdb.txt”,“4WF9_SA50S_teli.pdb.txt”, “SA50S_lefamulin.pdb.txt” and“SA50S_lincomycin.pdb.txt”, respectively.

According to some of any of the embodiments presented herein, the atomiccoordinates comprise atomic coordinates of at least one ribosomal RNA(rRNA) and further comprises atomic coordinate of at least one ribosomalprotein (rProtein).

In addition to the atomic coordinates and protein and RNA sequence dataof the large ribosomal subunit, the computer system may further includeprotein and RNA sequence and atomic coordinates of other ribosomalsubunits. For example, the data-storage device of the computer systeminclude the atomic coordinates and/or the sequence data of ribosomalsubunits from Staphylococcus aureus, Thermus thermophilus, Escherichiacoli, Haloarcula marismortui, Deinococcus radiodurans and Saccharomycescerevisiae, Tetrahymena thermophila.

In some of any of the embodiments of the present invention, the atomiccoordinates of D. radiodurans comprise at least a portion of the atomiccoordinates deposited at the Protein Data Bank under accession numberselected from the group consisting of PDB IDs: 2ZJR, 3DLL, 2OGM, 2OGN,2OGO, 1XNP, 1SM1, 1P9X, 1JZY and 4U67.

In some of any of the embodiments of the present invention, the atomiccoordinates of E. coli comprise at least a portion of the atomiccoordinates deposited at the Protein Data Bank under accession numberselected from the group consisting of PDB IDs: 2AW4, 3R8S, 3OAT and3OFR.

In some of the respective embodiments of the present invention, theatomic coordinates of T. thermophilus comprise at least a portion of theatomic coordinates deposited at the Protein Data Bank under accessionnumber selected from the group consisting of PDB IDs: 2WDL, 2WDK, 3OI3and 3OHD.

In some of the respective embodiments of the present invention, theatomic coordinates of H. marismortui comprise at least a portion of theatomic coordinates deposited at the Protein Data Bank under accessionnumber selected from the group consisting of PDB ID: 1S72, 3CC2 3CPW and1YIJ.

In some of the respective embodiments of the present invention, theatomic coordinates of T. thermophila comprise at least a portion of theatomic coordinates deposited at the Protein Data Bank under accessionnumber selected from the group consisting of PDB ID: 4A17, 4A18, 4A19,4A1A, 4A1B, 4A1C, 4A1D and 4A1E.

In some of the respective embodiments of the present invention, theatomic coordinates of S. cerevisiae comprise at least a portion of theatomic coordinates deposited at the Protein Data Bank under accessionnumber selected from the group consisting of PDB ID: 3U5B, 3U5C, 3U5D,3U5E, 3U5F, 3U5G, 3U5H and 3U5I.

Using sequence alignment and experimental structural information, thestructure of ribosomal particles which have not yet been obtainedexperimentally can be calculated computationally; hence, thedata-storage device of the computer system presented herein may furtherinclude the structure of a host of a pathogenic bacterium. A hostorganism can be a mammalian, thus the computationally-calculatedstructure of a ribosomal subunit can even be the structure of a humanribosome, or at least some subunits and/or parts thereof. Such astructure can be used to gain insights on the minute structuraldifferences between pathogenic and human host ribosome, allowing thedesign of de novo ligands which will be selective towards the pathogenicspecies and substantially inactive towards the host.

The atomic coordinates of any one of the SA50S native and complexstructures, include the atomic coordinates of at least one binding sitein the SA50S subunit. As used herein, the term “binding site” refers toa region on a protein, DNA, RNA or a combination thereof, to whichspecific molecules and/or ions (ligands) may bind to reversibly orirreversibly. As known in the art, ribosomal subunits comprise aplurality of binding sites which have been identified by variousexperimental methods including crystal structure elucidation, and someof those binding site have been identified or attributed to the largeribosomal subunit. It is noted that some binding site may be attributedto more than one subunit since these binding sites are defined by atomswhich belong to more than one subunit, e.g., a binding site which isfound at the interface between the small and large subunits.

According to some embodiments of the present invention, the term“binding site” refers to a locus in or on the ribosomal subunit which isrelated to one of more ribosomal functions in the sense that when beingoccupied by a ligand or freed thereof, the ribosomal function isaltered. A binding site which is associated with a ribosomal functionalis also referred to herein as a “ribofunctional locus”. According tosome embodiments, the term “ribofunctional locus” refers to a region ofthe ribosome or ribosomal subunit that participates, either actively orpassively, in decoding of the genetic information (translation), or inprotein or polypeptide synthesis within the ribosome or ribosomalsubunit and/or export or translocation of a protein or polypeptide outof a ribosome.

Exemplary binding sites, or ribofunctional loci, which are associatedwith the large ribosomal subunits, the atomic coordinates of at least aportion of which are part of the atomic coordinates of the largeribosomal subunit of a pathogenic bacterium excluding Escherichia coli,a pathogenic Gram positive bacterium, or a pathogenic bacteriumexhibiting a degree of 23S rRNA sequence identity of at least 80%compared to the 23S rRNA of Staphylococcus aureus, such as, e.g., SA50S,include without limitation an inter-subunit interface, a peptidyltransferase site, a GTPase center, an mRNA binding site, an A-site, aP-site, an E-site, a polypeptide exit tunnel, a translation initiationfactor (IF1) binding site, a translation initiation factor (IF2) bindingsite, a translation initiation factor (IF3) binding site, an elongationfactor G (EF-G) binding site, elongation factor Tu (EF-Tu) binding site,hibernation factor HPF binding site, hibernation factor RMF bindingsite, hibernation factor YfiA binding site, a GTP binding site and aricin binding site.

According to embodiments of some aspects of the present invention, thereis provided a computer readable medium comprising at least a portion ofthe positioning data in a retrievable format, which is indicative ofatomic coordinates determined from an electron density map calculatedfrom X-rays diffraction data obtained using a crystallized largeribosomal subunit of the pathogenic bacterium in thecomposition-of-matter provided herein.

As used herein, the term “computer readable medium” refers to any mediumwhich can be read and accessed directly by a computer. Such mediainclude, but are not limited to, a magnetic storage media, such asfloppy discs, hard disc storage medium, and magnetic tape; opticalstorage media such as optical discs or CD-ROM; electrical storage mediasuch as RAM and ROM; and hybrids of these categories such asmagnetic/optical storage media; a solid-state memory device; an acousticdata; a chemically or photochemically recorded data; and any othermachine-readable medium or automated data medium format known in theart. A skilled artisan can readily appreciate how any of the presentlyknown computer readable mediums can be used to create an articlecomprising computer readable medium having recorded thereon an aminoacid and/or nucleotide sequence, X-ray diffraction data, and/or atomiccoordinates of the present invention.

As used herein, the term “recorded” refers to any process for storinginformation on computer readable medium. A skilled artisan can readilyadopt any of the presently known methods for recording information oncomputer readable medium to generate articles comprising computerreadable medium having recorded thereon an amino acid or nucleotidesequence, atomic coordinates and/or X-ray diffraction data of thepresent invention.

The computer readable medium can further include, according to someembodiments of the present invention, the X-ray diffraction data as acollection of digitized images, a list of reflection indexes andcorresponding intensities and reflection spread, or any other format ofrecording the direct experimental results of the X-ray diffraction dataobtained from a crystal of a large ribosomal subunit of a pathogenicbacterium.

The computer readable medium can further include, according to someembodiments of the present invention, atomic coordinates and/orrProtein/rRNA sequence data of any other ribosomal subunit, obtainedeither experimentally or computationally.

The computer readable medium can further include, according to someembodiments of the present invention, atomic coordinates of any known orputative ligand of any ribosomal subunit, a putative ligand of a largeribosomal subunit of a pathogenic bacterium, a pathogenic Gram positivebacterium, or a pathogenic bacterium exhibiting a degree of 23S rRNAsequence identity of at least 80% compared to the 23S rRNA ofStaphylococcus aureus, such as, e.g., SA50S; while the putative ligandcan be obtained using computer-aided structure-based drug designmethodologies known in the art.

It is noted that comparative structural superimposition and sequencealignment comparisons and analysis, using the computer system providedherein and including the structure of a large ribosomal subunit of apathogenic bacterium provided herein, can be used, according toembodiments of the present invention, to find structural differencesbetween various ribosomal subunits, which can be exploited in the designof ligands that will exhibit species-specific affinity towards specificbinding sites.

Computer-Aided Structure-Based Drug Design:

According to some embodiments of the present invention, the atomiccoordinates of any ribosomal subunit, whether determined using X-raydiffraction, molecular modeling, homology modeling or molecularreplacement, as these terms are known in the art, may be used inrational drug design (RDD) to design de novo ligands which can be used,for example, as novel modulators, inducers, mimetics or inhibitors ofribosomal function, which generally relate to cellular proteinsynthesis. It is has been contemplated by the present inventors that byusing, for example, the SA50S subunit structure disclosed herein and theprinciples of RDD and computer-aided/assisted drug design (CADD), anordinary skilled artisan can design, prepare, test, refine and use denovo protein synthesis inhibitors specifically engineered to reduce,induce, disrupt, augment or otherwise affect cellular ribosomal functionin an organism or species of interest. For example, by using theprinciples discussed herein, the skilled artisan can engineer de novoligands that specifically target and inhibit ribosomal function in apathogenic bacterium, for example, a particular cocci bacterium, whilepreserving ribosomal function in a host, for example, a eukaryoticorganism, specifically a mammal, and more specifically, a human. As aresult, the atomic coordinates provided and discussed herein permit theskilled artisan to design de novo antibacterial agents that can killcertain pathogenic organisms while having little or no toxicity in theintended recipient, for example, a human, or to non-pathogenic bacteriathat reside in the host.

It has been contemplated that structure-based drug design proceduresusing atomic coordinates of the large ribosomal subunit, according toany embodiment of the present invention, including that of a pathogenicbacterium, a pathogenic Gram positive bacterium, or a pathogenicbacterium exhibiting a degree of 23S rRNA sequence identity of at least80% compared to the 23S rRNA of Staphylococcus aureus, such as, e.g.,SA50S subunit, can be facilitated most readily via CADD usingconventional computer hardware and software known and used in the artand discussed hereinabove.

It is noted that computer-aided drug design methods can utilizeexperimentally obtained positioning data of one target molecule, such asthe structure if the SA50S subunit, to design ligands for largeribosomal subunits of other species, particularly othergenetically-related bacteria, based on the non-limiting theory thattheir three-dimensional structure is similar due to a relatively highdegree of identity in their 23S rRNA sequence (see, “degree of 23S rRNAsequence identity” hereinabove). Hence, the computer-aided drug designprocedure can utilize the atomic coordinated of the SA50S subunit, asprovided herein.

The putative ligands may be designed de novo or may be designed as amodified variant of an already known (pre-existing) ligand, for example,a pre-existing antibacterial agent or a conjugate of one or morepre-existing ligands and/or fragments thereof. According to someembodiments of the present invention, pre-existing ligands orpre-existing antibacterial agents are ligands and antibacterial agentswhich have been pre-verified by in-vitro and/or in-vivo bioactivityassays and found to have an affinity to the ribosome or ribosomalsubunit of interest, using conventional methodologies. In some of any ofthe respective embodiments of the present invention, a putative ligandis having a molecular weight of less than about 1,500 grams/mol.

Once designed, putative ligands can be synthesized using standardmethodologies known and used in the art. Following synthesis, thecandidate molecules can be screened for bioactivity, for example, bytheir ability to reduce or inhibit ribosome function, their ability tointeract with or bind to a ribosome or a ribosomal subunit oncecontacted with the ribosome or the ribosomal subunit. Based in part uponthese results, the candidate molecules may be refined iteratively usingone or more of the foregoing steps to produce a ligand with a moredesirable biological activity. In general, a more desirable biologicalactivity typically refers to a ligand exhibiting a high affinity (strongbinding) and a high specificity and thus selective affinity towards aribosome of a particular species, allowing the use of the ligand as anantibiotic drug requiring clear distinction between the pathogen and themammalian host, low doses to eradicate a target pathogenic organismwhile exhibiting minimal adverse effects on the host organism.

The computer system provided herein may further include CADD programs,databases and other software components for calculating at least onestructure of a putative ligand for binding to at least one binding sitein, for example, the SA50S subunit. A computer-aided drug design programsuit may include general purpose molecular modeling programs such as,but not limited to structure alignment program for superimposing atomiccoordinates of independently-obtained structures; molecular mechanics,molecular dynamics and multifunctional programs which can be used tocalculate a molecular model of a ribosomal subunit even withoutexperimentally-obtained structural data; quantum chemistry calculationsfor small molecules; molecular orbital or quantum mechanicalcalculations; database of molecular structures of large and smallmolecules; software for storage and retrieval of molecular structuredata; molecular graphic software for large and small molecules; andprograms to visualize molecules.

Exemplary software for general purpose molecular modeling andcomputer-aided drug design include without limitation AMBER by PeterKollman and coworkers; Midas Plus by UCSF Computer Graphics Laboratory;CHARMM by Martin Karplus and coworkers; QUANTA/CHARMm by MolecularSimulations Inc. (MSI); Insight/DISCOVER by Biosym, Inc.; SYBYL andAlchemy III by Tripos, Inc.; ECEPP by Harold Scheraga and coworkers; MM3by Norman Allinger and coworkers; Chem3D Pro by CambridgeSoft Corp.;Desktop Molecular Modeller by Oxford Elec. Publishing; MolecularModeling Pro by WindowChem Software; and PC MODEL by Serena Software.

Additional computer programs useful for viewing or manipulatingthree-dimensional structures include: Midas (University of California,San Francisco); MOIL (University of Illinois); Yummie (Yale University);MacroModel (Columbia University); Cerius (Molecular Simulations, Inc.);LabVision (Tripos, Inc.); Rasmol (Glaxo Research and Development);Ribbon (University of Alabama); NAOMI (Oxford University); ExplorerEyechem (Silicon Graphics, Inc.); Univision (Cray Research); Molscript(Uppsala University); Chem-3D (Cambridge Scientific); Chain (BaylorCollege of Medicine); O (Uppsala University); GRASP (ColumbiaUniversity); X-Plor (Molecular Simulations, Inc.; Yale University);Spartan (Wavefunction, Inc.); Catalyst (Molecular Simulations, Inc.);Molcadd (Tripos, Inc.); VMD (University of Illinois/Beckman Institute);Sculpt (Interactive Simulations, Inc.); Procheck (Brookhaven NationalLibrary); DGEOM (QCPE); RE_VIEW (Brunell University); Modeller (BirbeckCollege, University of London); Xmol (Minnesota Supercomputing Center);Protein Expert (Cambridge Scientific); HyperChem (Hypercube); MD Display(University of Washington); PKB (National Center for BiotechnologyInformation, NIH); ChemX (Chemical Design, Ltd.); Cameleon (OxfordMolecular, Inc.); and Iditis (Oxford Molecular, Inc.).

Exemplary databases that may be used in the computer-aided drug designbased on the structure of a large ribosomal subunit of a pathogenicbacterium, such as the SA50S subunit structure presented herein,include, without limitation, structural data of various homologousribosomal subunits obtained from X-ray diffraction, NMR and otherstructure determination procedures; protein and nucleic acid sequencedata; quantitative structure-activity relationship (QSAR of conventionalcompound synthesis and combinatorial chemistry) and other chemical data.

The computer-aided drug design based on the structure of a largeribosomal subunit of a pathogenic bacterium presented herein, such asthe SA50S subunit structure, may further include the use of molecularenergy parameters pertaining to structural geometry and folding,molecular dynamics parameters pertaining to conformational changes, andmolecular recognition parameters pertaining to putative ligand design.

The computer-aided drug design based on the availability of atomiccoordinated (structure) of, for example, the SA50S subunit presentedherein may further include the use of de novo ligand design methodswhich include, without limitation, fragment location methods, site pointconnection methods, fragment connection methods, sequential buildupmethods, whole molecule methods, random connection methods and the likescomputational procedures known in the art.

Determining desirable locations of atoms or small fragments within abinding site on SA50S in the computer-aided drug design based on theSA50S subunit structure presented herein may be accomplished by usingGRID by Goodford and coworkers; GREEN by Itai and coworkers; HINT byKellogg and coworkers; Dean by Lewis and coworkers; CAVEAT by Bartettand coworkers; and HOOK by Hubbard and coworkers.

The computer-aided drug design based on the SA50S subunit structurepresented herein may further include the use of molecular surfacematching (docking) and binding scope evaluation methods, which are aimedat fitting known compounds into a binding site in various orientations,assessing shape and/or electrostatic complementarity and other chemicaland physical parameters. Such methods are implemented in software suitswhich include, without limitation, Dock by Kuntz and coworkers; AUTODOCKby Olsen and coworkers; and various Monte Carlo approaches.

The computer-aided drug design based on the SA50S subunit structurepresented herein may make use of general purpose CADD suites includingcombinatorial chemistry software for structural accuracy and specificityand for rapid synthesis procedures and structural diversity.

Hence, according to some embodiments of the present invention, thedata-storage device(s) in computer system may further include the atomiccoordinates of a putative ligand, which has been designed for binding toone of the binding sites in the large ribosomal subunit, wherein theputative ligand is a product of a computer-aided drug design procedurebeing a structure-based drug design, namely the putative ligand isdesigned based on a structure of at least one binding site in the largeribosomal subunit of a pathogenic bacterium, such as the presentlyprovided SA50S subunit.

Identifying Ligands of Ribosomal Subunits:

The tools and methodologies provided herein may be used to identifyand/or design ligands which bind and/or interact in desirable ways withribosomes and ribosomal subunits. Basically, the procedures utilize aniterative process whereby the ligands are designed, prepared, tested andcharacterized. De novo ligands can be designed based on the informationgained in the testing and characterization of the initial molecules andthen such newly identified molecules can themselves be tested andcharacterized. This series of processes may be repeated as many times asnecessary to obtain molecules with desirable binding properties and/orbiological activities. Methods for identifying candidate molecules arediscussed in more detail below.

According to some embodiments of an aspect of the present invention,there is provided a method for designing and selecting a putative ligandhaving an affinity to a binding site of a large ribosomal subunit of apathogenic bacterium excluding Escherichia coli, a pathogenic Grampositive bacterium, or a pathogenic bacterium exhibiting a degree of 23SrRNA sequence identity of at least 80% compared to the 23S rRNA ofStaphylococcus aureus, such as the large ribosomal subunit ofStaphylococcus aureus. According to some embodiments, the ligandexhibits high affinity and high specificity to the ribosomal subunit.

The method can be implemented using a computer system that includes theatomic coordinates (positioning data or structure) of a large ribosomalsubunit of a pathogenic bacterium excluding Escherichia coli, apathogenic Gram positive bacterium, or a pathogenic bacterium exhibitinga degree of 23S rRNA sequence identity of at least 80% compared to the23S rRNA of Staphylococcus aureus, such as the SA50S subunit, andsequence data and software as described hereinabove. According to someembodiments, the design of a putative ligand can be facilitated usingconventional computer systems available commercially from, for example,Silicon Graphics Inc. and Sun Microsystems, running, for example, UNIXbased, Windows NT on IBM OS/2 operating systems, and capable of runningconventional computer programs for molecular modeling and rational drugdesign. It is noted herein that any computer system having the overallcharacteristics of a typical computer system may be useful in thepractice of the invention. Specifically, a typical computer systemtypically include input, processing, storage and output components inelectrical communication with one another via, for example, an internalbus or external network, a central processing unit, a random accessmemory (RAM), a read only memory (ROM), a monitor or terminal, andoptimally an external or internal data-storage device, for example, adiskette, CD ROM, or magnetic tape or a solid-state memory device. Adecentralized (cloud based) computer system is also contemplated withinthe scope of the present invention.

According to some embodiments of the present invention, a putativeligand that can bind to a ribosomal subunit of a pathogen at a desiredaffinity and thus inhibit its protein biosynthesis can be designedentirely de novo or may be based upon a pre-existing (pre-verified)protein biosynthesis inhibitor(s). Either of these approaches can befacilitated by computationally screening databases and libraries ofsmall molecules for chemical entities, agents, ligands, or compoundsthat can bind in whole, or in part, to ribosomes and ribosomal subunits,more preferably to large ribosomal subunits, more preferably to largeribosomal subunits of pathogenic bacterium excluding Escherichia coli, apathogenic Gram positive bacterium, or a pathogenic bacterium exhibitinga degree of 23S rRNA sequence identity of at least 80% compared to the23S rRNA of Staphylococcus aureus, and more preferably to SA50Sribosomal subunit. In this screening, the quality of fit of suchentities or compounds to the binding site or sites may be judged eitherby shape complementarity or by estimated interaction energy.

Briefly, the method comprises:

(a) obtaining positioning data indicative of atomic coordinates of atleast one binding site of the large ribosomal subunit of a pathogenicbacterium, as defined herein, such as the SA50S subunit as providedherein;

(b) calculating the molecular surface of the binding site whiledenoting, for example, solvent accessible surface, chargedfunctionalities and groups, hydrophobicity, hydrogen-bonddonors/acceptors, and other chemical and physical characteristics of themolecular surface, using a suitable computer program;

(c) computationally constructing at least one chemically feasible ligandhaving a molecular surface that matches the molecular surface of abinding site of choice, namely a ligand exhibiting a pharmacophorecorresponding to a binding site of choice, using suitable computerprograms that correlate the molecular surface attributes provided instep (b) with feasible and matching chemical scaffolds and functionalgroups in the ligand.

In some of the respective embodiments of the present invention, themethod further comprises computationally constructing a library ofstructures of chemically feasible ligands having a molecular surfacethat matches the molecular surface of the binding site (each exhibitinga pharmacophore corresponding to a binding site of choice):

(d) computationally determining a matching/binding score for each of thedesigned ligands, using docking algorithms that account for energyminimization, structural geometry constrains, shape and/or electrostaticcomplementarity and the likes; and

(e) based on the matching score, selecting at least one putative ligandhaving a desired affinity to the molecular surface of the binding siteof the large ribosomal subunit of a pathogenic bacterium.

According to some embodiments of the present invention, characterizingthe binding of a ligand of the ribosomal subunit comprises obtaining orsynthesizing a ligand, contacting the ligand with the ribosomal subunitthereby forming a complex thereof, and analyzing the complex by X-raycrystallography.

In some of any of the respective embodiments of the present invention,the search for a ligand of the large ribosomal subunit of a pathogenicbacterium can be based on a computerized search for complementarity ofmembers of a library of known ligand structures. Such a search mayprovide insights for further manipulations of the known ligands, as wellas serve as an advantageous starting model for de novo designed ligands.According to some embodiments, the library may include variouschemically feasible variants of pre-existing ligands, namely a set ofligands which are based on a known ligand which has been modifiedcomputationally while observing feasible chemistry constrains and whileallowing a wide range of chemically diverse variations.

According to some embodiments, a set of known and pre-verified ligandsand variants thereof undergo the procedure of Step (d) as the de novodesigned ligands of Step (c).

The procedure for computationally designing, docking and scoring thebinding affinity of putative de novo designed ligands and/or modifiedligands and/or modified known ligands, as described herein, can berepeated, according to some embodiments, using a molecular surface ofthe same binding site in a ribosomal subunit of another species, such asa host or a benign (non-pathogenic) species. A high affinity to amolecular surface can be seen as a match between the ligand and thetarget at that binding site, and a lower affinity to a molecular surfacecan be seen as a mismatch between the ligand and the target at thatbinding site. Such a procedure provides a list of putative ligands (alibrary) which exhibit a species-specific binding affinity, namely,ligands may be identified also according to a higher affinity to theribosomal subunit of a pathogenic and a relatively lower affinity to theribosomal subunit of the other organism.

A list of non-limiting examples of antibiotic binding sites in the SA50Sis provided in the Examples section that follows below. A list ofnon-limiting examples of regions where structural differences have beenidentified between the SA50S and the corresponding regions in otherribosomal subunits is provided in the Examples section that followsbelow. The skilled artisan in possession of the foregoing or otherexemplary binding sites may use the method for designing a ligand of theSA50S subunit provided herein to identify ligands that potentially bindto one or more of the binding sites and/or inhibit ribosomal activity.Furthermore, by taking into account which of the residues that definethe target site are conserved in terms of sequence similarity betweenpathogens but not conserved between host species, the skilled artisancan design new species-specific protein synthesis inhibitors.

As known in the art, ribosomes from bacteria, archaea and eukaryotesdiffer in their size, sequence, structure, and the ratio of protein toRNA. The differences in structure allow some antibiotics to killbacteria by inhibiting functions (e.g., protein synthesis) of theirribosomes, while leaving eukaryotic ribosomes (e.g., those of a hostorganism such as a human) unaffected. It is appreciated that the skilledartisan can take advantage of the regions that are not conserved betweena bacterial pathogen and a eukaryotic host organism to provide targetregions for rational drug design. By way of example, FIG. 14A showscertain regions of the erythromycin tunnel entrance binding pocket thatare conserved between Staphylococcus aureus and S. cerevisiae andregions of the same pocket that are not conserved between Staphylococcusaureus and S. cerevisiae, where S. cerevisiae serves as a representativeof a eukaryotic organism. In addition, the skilled artisan when inpossession of mutations that prevent or reduce antibiotic activity(i.e., are related to antibiotic resistance) can use this information tomodel the relevant antibiotic binding product which can then be used asa basis for rational drug design to identify small molecules, having,for example, a molecular weight of less than about 1,500 grams/mol, thatovercome drug resistance. It is contemplated that a variety of computermodeling procedures, for example, homology modeling protocols, can beused to provide a model of a drug resistance target site by implementingsite directed mutagenesis of nucleotides and/or amino acids and thenusing the appropriate energy minimization and refinement protocols.

Starting with the structure of the exemplary SA50S ribosomal subunitprovided herein, as well as other known structure of the large ribosomalsubunit of other species, the structure of the ribosome from anon-targeted organism (for example, the human 60S ribosomal subunit) canbe constructed by homology modeling, i.e., by changing the structure ofresidues at a target site of interest for the residues at the samepositions in of the non-target ribosome. This can be achieved byremoving computationally the side chains from the ribosome of knownstructure and replacing them with the side chains of the unknownstructure put in sterically plausible and chemically feasible positions.In this way, it can be understood how the shapes of the target siteswithin the targeted and non-targeted ribosomes differ. This process,therefore, provides information concerning how a molecule that binds thetarget site can be chemically altered in order to produce ligands thatbind tightly and specifically to the targeted ribosome but willsimultaneously be prevented from binding to the non-targeted ribosome.

Accordingly, knowledge of portions of the bound ligands that face thesolvent permits introduction of other functional groups for additionalpharmaceutical purposes. The process of homology structure modeling canalso be used to understand the mechanisms whereby mutant ribosomesbecome resistant to the effects of pharmaceuticals. Furthermore, withknowledge of the regions in a large ribosomal subunit of a pathogenicbacterium that participates in drug resistance, the skilled artisan maydesign new ligands that overcome the problem of drug resistance.

The use of homology structure modeling to design ligands that bind moretightly to the target ribosome than to the non-target ribosome haswide-spread applicability. The methods outlined herein can be used tocontrol any targeted organism, for example, a pathogenic bacterium, bydesigning molecules that inhibit large ribosomal subunits of thetargeted organisms while failing to inhibit the 50S or 60S ribosomalsubunit of the non-targeted organism, for example, a host, to the sameextent or not at all. The molecules identified or prepared by themethods of the present invention can be used to control the targetedorganisms while causing the non-targeted organism little or no adverseeffects. Thus, the ligands, identified or designed using the methodsaccording to embodiments of the present invention, can be designed sothat their administration kills the target organisms or inhibits someaspect of the biological functions of the target organisms while failingto have a similar effect on the non-targeted organism. The adverseeffects of the agent on the targeted organisms may include, but are notlimited to, death of the target organism; slowing growth rates; slowingor eliminating passage from one growth phase to another (e.g., extendingthe larval growth stage); slowing or eliminating reproduction,decreasing or preventing mating, decreasing or eliminating offspringproduction, limiting or eliminating target organism weight gains;decreasing or eliminating feeding ability and behaviors; and disruptingcellular, tissue and/or organ functions.

The novel ligands contemplated according to some embodiments of thepresent invention can be useful as antimicrobial agents (e.g.,antifungals, antibacterials, antiprotozoals, etc.) to target specificorganisms. For example, the novel ligands can target animal, prokaryoticorganisms (pathogenic bacteria), and eukaryotic multicellular pests.

Antimicrobial agents that inhibit protein synthesis by interacting withribosomes are known to the skilled artisan. A few examples are discussedbelow. These known agents can be modified to obtain novel agents byusing computer modeling techniques and knowledge of the structure ofribosomes and ribosomal subunits and the structure of ribosome/agent andribosomal subunit/agent complexes, such as the native and complexstructures of large ribosomal subunits of a pathogenic bacteria providedherein, such as the SA50S.

The design of ligands of ribosomes or ribosomal subunits according toembodiments of the present invention generally involves consideration oftwo factors: the ligand is to be capable of physically and structurallyassociating with the large ribosomal subunit, while consideringnon-covalent molecular interactions which are present in the associationof ribosomes and ribosomal subunits with the ligand, includingelectrostatic interactions, hydrogen bonding, van der Waals andhydrophobic interactions; and the ligand is to be able to assume aconformation that allows it to associate with the ribosomes or ribosomalsubunits, more preferably to large ribosomal subunits of pathogenicbacterium excluding Escherichia coli, a pathogenic Gram positivebacterium, or a pathogenic bacterium exhibiting a degree of 23S rRNAsequence identity of at least 80% compared to the 23S rRNA ofStaphylococcus aureus, and more preferably with the SA50S ribosomalsubunit. Although certain portions of the molecule may not directlyparticipate in this association with a ribosome or ribosomal subunitsthose portions may still influence the overall conformation of themolecule. This, in turn, may have an impact on binding affinities,therapeutic efficacy, drug-like qualities, and potency. Suchconformational requirements include the overall three-dimensionalstructure and orientation of the ligand in relation to all or a portionof the binding site or other region of the ribosomes or ribosomalsubunits, or the spacing between functional groups of a ligandcomprising several functional groups that directly interact with theribosomes or ribosomal subunits, more preferably to large ribosomalsubunits of pathogenic bacterium excluding Escherichia coli, apathogenic Gram positive bacterium, or a pathogenic bacterium exhibitinga degree of 23S rRNA sequence identity of at least 80% compared to the23S rRNA of Staphylococcus aureus, and more preferably with the SA50Sribosomal subunit.

The putative calculated inhibitory or binding effect of a ligand onribosomes and ribosomal subunits may be analyzed prior to its actualsynthesis and testing by the use of computer modeling techniques. If thetheoretical structure of the given ligand suggests insufficientinteraction and association between it and ribosomes or ribosomalsubunits, synthesis and testing of the ligand is obviated. However, ifcomputer modeling indicates a strong interaction, the ligand may then besynthesized and tested for its ability to interact with the ribosomes orribosomal subunits and inhibit protein synthesis. In this manner,synthesis of inoperative ligands may be avoided. In some cases, inactiveligands are synthesized, predicted on modeling and then tested todevelop a SAR (structure-activity relationship) for ligands interactingwith a specific region of the ribosome or ribosomal subunit, morepreferably to large ribosomal subunits of pathogenic bacterium excludingEscherichia coli, a pathogenic Gram positive bacterium, or a pathogenicbacterium exhibiting a degree of 23S rRNA sequence identity of at least80% compared to the 23S rRNA of Staphylococcus aureus, and morepreferably of the SA50S ribosomal subunit. As used herein, the term“SAR”, shall collectively refer to the structure-activity/structureproperty relationships pertaining to the relationship(s) between aligand's activity/properties and its chemical structure.

One skilled in the art may use one of several methods to identifychemical moieties or entities, ligands, or other agents for theirability to associate with a preselected target site within a ribosomesor ribosomal subunit. This process may begin by visual inspection orcomputer assisted modeling of, for example, the target site on thecomputer screen based on the atomic coordinates of the SA50S ribosomalsubunit and/or its complexes with other analogues and antibacterialagents. In some of the respective embodiments of the present invention,ligand design uses computer modeling programs which calculate howdifferent ligands interact with the various binding sites of theribosome, ribosomal subunit, or a fragment thereof. Selected chemicalmoieties or entities, ligands, or agents may then be positioned in avariety of orientations, or docked, within at least a portion of thebinding site of a ribosome or ribosomal subunit, more preferably tolarge ribosomal subunits of pathogenic bacterium excluding Escherichiacoli, a pathogenic Gram positive bacterium, or a pathogenic bacteriumexhibiting a degree of 23S rRNA sequence identity of at least 80%compared to the 23S rRNA of Staphylococcus aureus, and more preferablyof a SA50S ribosomal subunit. Databases of chemical structures areavailable from, for example, Cambridge Crystallographic Data Center(Cambridge, U.K.) and Chemical Abstracts Service (Columbus, Ohio).Docking may be accomplished using software such as Quanta and Sybyl,followed by energy minimization and molecular dynamics with standardmolecular mechanics forcefields, such as CHARMM and AMBER.

According to some embodiments of the present invention, the affinity ofa designed ligand may be increased, compared to, for example, a templateligand, by designing a ligand so as to exhibit more functional groupsand moieties on its molecular surface, compared to the template ligand,which compliment and thus interact with counter functional groups on themolecular surface in or near a specific ribofunctional locus in thetarget ribosome; in other words, a ligand which exhibits a pharmacophorewhich better compliments the ribofunctional locus of interest in thetarget ribosome.

The specificity of the ligand towards a particular species is affordedby designing a ligand having functional groups and moieties that matchcounter functional groups in the ribosome of a target species that arespecific to that ribosome, namely the ligand is designed based onstructural differences between ribosomes of different species.Species-specific structural information is provided, for example, by theSA50S native and complex structures provided and described herein, whichis used to identify the SA-specific pharmacophores which are the mostdiverse compared to equivalent pharmacophores which bind, for example,to a host's ribosome.

As an alternative approach to the de novo structure-based drug designapproach, it is contemplated that pre-existing (pre-verified) ligands ofthe target pathogenic ribosome or ribosomal subunit may be used as astructural starting point (template) for the design of a novel ligand.It is contemplated that knowledge of the spatial relationship between apre-verified ligand, such as a known protein biosynthesis inhibitor, andits respective binding site within a ribosome (its ribofunctional locus)of a target pathogen is conducive to the identification of a moreelaborate pharmacophore, and to the design of a modified ligand that mayhave improved binding properties, for example, higher binding affinityand/or higher species specificity, relative to the original (template)ligand it has been derived from. Alternatively, structural knowledge ofmore than one pre-verified ligands and their respective ribofunctionalloci in the ribosome or ribosomal subunit of the target pathogenicmicroorganism is conducive to the design of a novel ligand thatcontains, for example, a portion of each of the pre-verified ligands.Such a ligand is referred to herein as adduct or conjugate.

The positioning data of each pre-verified ligand relative to the largeribosomal subunit of a pathogenic bacterium, such as the SA50S providedherein, provides information on what portions of the ribosome orribosomal subunit and what portions the ligand are in contact.Accordingly, using this structural information obtained experimentally,the skilled artisan can identify ribofunctional loci that can be usedfor de novo drug design, as discussed above, as well as identifyportions of a ligand that can act as a ribosome binding moieties.

Based on the structural information pertaining to a large ribosomalsubunit of a pathogenic bacterium, such as the SA50S provided herein,the skilled artisan may readily identify a more elaborate pharmacophoreand design novel adduct ligands that comprise a ribosome binding moietyof a first pre-existing ligand or an analogue or a derivative thereofand a ribosome binding moiety of a second, different pre-existing ligandor an analogue or a derivative thereof, each expanding the pharmacophoreof the other. As stated hereinabove, in some of any of the respectiveembodiments of the present invention, a pre-existing ligand used astemplates for the expansion of the known pharmacophore and to the designof a novel adduct ligand, may refer to a pre-verified antibacterialagent. The resulting novel designed adduct ligand, according toembodiments of the present invention, preferably binds simultaneously toeach of the respective ribofunctional loci within the ribosomal subunitby exhibiting a more elaborate pharmacophore, and thus exhibits higherbinding affinity and/or higher specificity to the ribosomal subunitcompared to the affinity and/or specificity exhibited by the twotemplate antibacterial agents individually, and even exhibit synergisticbinding properties compared to those of the two individual templateantibacterial agents.

It is noted herein that embodiments of the present invention are meantto encompass design of novel ligands based on combining thepharmacophores of more than two, more than three and more than fourpre-existing ligands; namely the expansion of the known pharmacophore iseffected by obtaining complex structures of a ribosome or a ribosomalsubunit with more than two different pre-existing ligands bound thereto,and the resulting novel ligand may share moieties (pharmacophoreelements) stemming from more than two pre-existing ligands. In general,any number of complex structures of a ribosomal subunit bound to apre-existing ligand can be used as a training set for the procedure ofidentification and expansion of a pharmacophore pertaining to any givenribofunctional locus, wherein the training set is the collection ofligands bound in a given binding site or near it, which can besuperimposed on one-another based on common target's atoms positions.

The positioning data of the exemplary SA50S provided herein, nativeand/or complexes thereof with pre-verified antibacterial agents, allowthe skilled artisan to identify putative antibacterial agents that maybe used as templates in the synthesis of novel adducts, and also providestructural information necessary to produce linking moieties such thateach ribosome binding moiety in the adduct is properly positioned andorientated relative to its respective binding site. As a result, it iscontemplated that the skilled artisan may produce adduct ligands (adductantibacterial agents) that bind to a SA ribosome or ribosomal subunitwith a higher affinity and specificity, and/or exhibit higherribofunctional inhibitory activity than either of the individualtemplate antibacterial agents used to design the adduct. Alternatively,the conjugate (adduct antibacterial agent) may overcome resistancephenotypes that may have developed against either of the templateantibiotics.

As used herein, the phrase “linking moiety” describes a chemical moietythat links two other chemical moieties via one or more covalent bonds,hence linking therebetween. In general, the linking moiety can be formedduring a chemical reaction, such that by reacting two or more reactivegroups, the linking moiety is formed as a new chemical entity which cancomprise a bond (between two atoms), or one or more bonded atoms.Alternatively, the linking moiety can be an independent chemical moietycomprising two or more reactive groups to which compatible reactivegroups on other compounds can be attached, either directly orindirectly. In the context of the present embodiments, the linkingmoiety can be, for a non-limiting example, a single, double or triplecovalent bond; a heteroatom (such as, but not limited to, O, N, S, andP); and a saturated or unsaturated, aliphatic or aromatic, substitutedor unsubstituted hydrocarbon chain having 1-6 carbon atoms, optionallyinterrupted by 1-6 heteroatoms.

For example, the proximity of a site occupied by the hydroxyl group ofthe tricyclic mutilin core of BC-3205 to the site occupied by thedesosamine sugar of telithromycin and the proximity of a site occupiedby the valyl moiety NH₂ of BC-3205 to the site occupied by thealkyl-aryl arm of telithromycin suggests that adducts comprising theaforementioned hydroxyl portion of both BC-3205 and the desosamine sugarportion of the telithromycin, spaced and positioned relative toone-another as they are seen in the crystal structure of thecorresponding complex, may be an effective inhibitor of proteinsynthesis of the exemplary SA50S.

Furthermore, the positioning data of the SA50S provided herein, nativeand complexes, allow the skilled artisan to use the informationpertaining to identify ribosome binding moieties and design other typesof ribofunctional inhibitors, also for large ribosomal subunit of otherpathogenic bacteria. For example, with an understanding of the ribosomecontact region and the surrounding environment, and the identificationof species-specific variations in the ribosome structure, the skilledartisan can design novel ligands, a portion of which is based upon theantibiotic binding site and another portion of which can be designed asa novel site that sterically inhibits or disrupts protein biosynthesiswithin the ribosome or secretion through the polypeptide exit tunnel.For example, the skilled artisan may combine the ribosome binding siteof pre-verified antibacterial agent or an analog or derivative thereof,which binds to one side of the polypeptide exit tunnel close to thepeptidyl transferase site, with, for example, a novel chemical moietywhich is not present in antibacterial agent, and which is identifiedbased on the structure of the SA50S, that can effectively block thepolypeptide exit tunnel. Furthermore, it is contemplated that theskilled artisan may take advantage of one or more of the many of theantibiotic binding sites known in the art, and those that can beelucidated based on the provisions disclosed herein to design entirelynew binding moieties.

According to some embodiments of the present invention, the skilledartisan can design ribofunctional ligands, for example, selectiveprotein synthesis inhibitors, that are tailored to be more potent withrespect to ribosomes of a target pathogenic organism, such as SA, andless potent, i.e., less toxic, to ribosomes of a non-target organism,for example, a host organism such as a human. The presently providedpositioning data of the SA50S, native and complexes thereof withpre-verified ligands, allow the skilled artisan to design modificationsto lead compounds, such as an antibacterial agent, that will bind moretightly to the SA50S ribosomal subunit and less tightly to anon-targeted ribosome, e.g., human 60S ribosomal subunit or a humanmitochondrial ribosome. The positioning data provided herein can also beused to guide the modification of pre-verified ligand to produce newligands that have other desirable properties for the applicableindustrial and other uses (e.g., as pharmaceuticals), such as chemicalstability, solubility or membrane permeability.

A variety of pre-verified ligands of the large ribosomal subunit thatdisrupt protein synthesis include, without limitation, chloramphenicols,macrolides, lincosamides, streptogramins, althiomycins, oxazolidinones,nucleotide analogs, thiostreptons (including micrococcin family),peptides, glutarimides, trichothecenes, TAN-1057, pleuromutilins,hygromycins, betacins, eveminomicins, boxazomycins, and fusidanes.

Members of the chloramphenicol family include, for example,Chloramphenicol and lodoamphenicol. Members of the macrolide familyinclude, for example, Biaxin (Clarithromycin), Zithromax (Azithromycin;azalide), Ketek (Telithromycin; ketolide), ABT-773, Tylosin, SpiramycinI, Spiramycin II, Spiramycin III, Erythromycin A, Carbomycin A,Telithromycin, Methymycin, Narbomycin, Lankamycin, Oleandomycin,Megalomycin, Chalcomycin, Niddamycin, Leucomycin, Angolamycin,Picromycin, and Relomycin. Members of the licosamide family include, forexample, Clindamycin and Lincomycin. Members of the streptogramin familyinclude, for example, Streptogramin A, Streptogramin B, Ostreogrycin G,Synercid, Virginiamycin S1, Virginiamycin S2, Virginiamycin S3,Virginiamycin S4, Vernamycin B, Vernamycin C, Patricin A, and PatricinB. A member of the althiomycin family includes, for example,Althiomycin. Members of the oxazolidinone family, include, for example,Linezolid, Eperezolid, and DuP721. Members of the family of nucleotideanalogs include, for example, Sparsomycin, Puromycin, Anisomycin, andBlasticidin S. Members of the thiostrepton family include, for example,Thiostrepton, Siomycin, Sporangiomycin, Micrococcin M1, Micrococcin P,and Thiopeptin. Members of the peptide family include, for example,Viomycin, Capreomycin IA, Capreomycin IB, Capreomycin IIA, andCapreomycin IIB. Members of the glutarimide family include, for example,Cycloheximide, Streptovitacins, Streptimidone, Inactone, Actiphenol.Members of the trichothecene family include, for example, Trichodermin,Trichodermol, Trichodermone, Vomitoxin, T-2 toxin, Trichothecin,Nivalenol, and Verrucarin A. Tan-1057 includes Tan-1057A, Tan-1057B,Tan-1057C, and Tan-1057D. Pleuromutilins include, for example,Pleuromutilin, Tiamulin, Azamilin, and Valnemulin. Hygromycins includesthe Hygromycin A antibiotics. Betacins include, for example, the familyof betacin natural products and VCR4219. Everninomicins include, forexample, Ziracin, Avilamycin, Evernimicin, and Curamicin. Boxazomycinsinclude, for example, Boxazomycin A and B. Fusidanes include, forexample, fusidic acid and 175, 20S-dihydrofusidic acid diethylene glycolhydrate.

Once a putative ligand has been designed or selected by the abovemethods, the affinity with which that ligand binds to the ribosome orribosomal subunit may be tested and optimized by computationalevaluation and/or by testing biological activity after synthesizing thecompound and contacting the same with the ribosome or ribosomal subunitof choice. Putative ligands may interact with the ribosomes or ribosomalsubunits in more than one conformation, each of which has a similaroverall binding energy. In those cases, the deformation energy ofbinding may be considered to be the difference between the energy of thefree molecule and the average energy of the conformations observed whenthe ligand binds to the ribosomes or ribosomal subunits, or to the largeribosomal subunits, or to the 50S ribosomal subunits of variouspathogenic bacteria, or to large ribosomal subunits of pathogenicbacterium excluding Escherichia coli, a pathogenic Gram positivebacterium, or a pathogenic bacterium exhibiting a degree of 23S rRNAsequence identity of at least 80% compared to the 23S rRNA ofStaphylococcus aureus, or to the SA50S ribosomal subunit.

A ligand designed or selected as binding to a ribosome or ribosomalsubunit may be further computationally optimized so that in its boundstate it preferably lacks repulsive electrostatic interaction with thetarget region. Such non-complementary (e.g., electrostatic) interactionsinclude repulsive charge-charge, dipole-dipole and charge-dipoleinteractions. Specifically, the sum of all electrostatic interactionsbetween the ligand and the molecular surface of its binding site whenthe ligand is bound to the ribosome or the ribosomal subunit, preferablymakes a neutral or favorable contribution to the enthalpy of binding.Weak binding ligands can also be designed by these methods so as toprovide SAR information.

Specific computer programs that can evaluate a ligand deformation energyand electrostatic interaction are available in the art. Examples ofsuitable programs include, without limitation, Gaussian 92, revision C(M. J. Frisch, Gaussian, Inc., Pittsburgh, Pa.); AMBER, version 4.0 (P.A. Kollman, University of California at San Francisco, Calif.);QUANTA/CHARMM (Molecular Simulations, Inc., Burlington, Mass.); OPLS-AA(“OPLS Force Fields.” W. L. Jorgensen. Encyclopedia of ComputationalChemistry, Schleyer, Ed.; Wiley: New York, 1998; Vol. 3, pp 1986-1989.)and Insight II/Discover (Biosysm Technologies Inc., San Diego, Calif.).These programs may be implemented, for instance, using a SiliconGraphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550.Other suitable computer systems and software packages are known to thoseskilled in the art.

Once a ligand has been selected or designed, as described above,substitutions may then be made in some of its atoms or side groups inorder to improve or modify its binding properties. Generally, initialsubstitutions are conservative, i.e., the replacement group willapproximate the same size, shape, hydrophobicity and charge as theoriginal group. It is to be understood that components known in the artto alter conformation and non-feasible chemical structures should beavoided. Such substituted ligands may then be analyzed for efficiency offit to the ribosome or ribosomal subunit by the same computer methodsdescribed herein.

The designed ligand can be prepared by chemical procedures known in theart, and can further be prepared according to a computer-assistedorganic synthesis (CAOS) program. CAOS programs are used in organicchemistry and computational chemistry to facilitate the task ofreactions design and prediction. A CAOS program can take into synthesisconsiderations the spatial limitations and constrains stemming from thestructure-based drug design process so as to suggest a syntheticprocedure that can afford a viable and chemically feasible ligand. Atypical CAOS program identifies a sequence of chemical reactions capableof producing a desired target molecule. CAOS algorithms typicallyutilize a database of known chemical reactions and a second database ofknown starting materials (i.e., typically commercially availablemolecules). Preferred synthetic plans and procedures are selectedaccording to cost considerations, expected yield, and avoidance ofhazardous reactants, reactions and intermediates. Exemplary CAOSprograms and software packages that can be used in the context ofembodiments of the present invention include, without limitation, WODCA,OSET, CHIRON, SynGen, LHASA, SYLVIA and ARChem.

In addition, the putative ribosome ligands, complexes or mimeticsthereof may be co-crystallized with ribosomes or their subunits andanalyzed using X-ray diffraction. The diffraction data are similarlyused to calculate the three-dimensional interaction of a putative ligandand the ribosome, ribosomal subunit, or a mimetic, in order to confirmthat the ligand binds to, or changes the conformation of, a particularbinding site on the ribosome or ribosomal subunit, or where the mimetichas a similar three-dimensional structure to that of a ribosome,ribosomal subunit or a fragment thereof.

A High-Affinity/High-Specificity Ligand:

Embodiments of the present invention provides the use of molecular andcomputer modeling techniques to design and select novel ligands ofribosomal particles, such as antibacterial agents or other therapeuticagents, that interact with ribosomes and ribosomal subunits. Suchantibiotics and other types of therapeutic agents include, but are notlimited to, antifungals, antivirals, antibacterials and the likes. It isnoted that the novel ligand, according to embodiments of the presentinvention, is designed to be structurally different from all knownand/or naturally occurring ligands, and further designed to exhibitimproved binding properties (affinity and specificity) compared to knownand/or naturally occurring ligands. Hence, previously known ligands ofthe ribosome of pathogenic bacteria, including all previously knownnaturally occurring and/or synthetic antibiotics, polypeptides, nucleicacids and the likes, are excluded from the scope of the presentinvention in the context of some embodiments thereof pertaining tode-novo designed ligands of the ribosome of the pathogenic bacteria.

It will be appreciated that due to the tight correlation betweenstructure and activity of biological molecular systems, and the tightcorrelation between biological molecular target structure and thespecificity and affinity of a given ligand, knowledge of the intricaterelations between the ribosomal subunit of one species and itscorresponding ligands can serve for insights for designing ligands thatwill be active (exhibit high affinity and specificity) with respect tocorresponding ribosomal subunit of variousphylogenetically/evolutionary/genealogically related species, as thisconcept is defined herein.

As described hereinabove, a ligand can be designed to exhibit anaffinity to a molecular surface of at least one binding site or aribofunctional locus of a ribosomal subunit of a pathogenic bacterium, apathogenic Gram positive bacterium, or a pathogenic bacterium exhibitinga degree of 23S rRNA sequence identity of at least 80% compared to the23S rRNA of Staphylococcus aureus, such as the SA50S provided herein.

According to an aspect of some embodiments of the present invention,there is provided a ligand having an affinity to a molecular surface ofat least a portion of a binding site or a ribofunctional locus of alarge ribosomal subunit of a pathogenic bacterium provided herein,designed by the method provided herein.

The general concept of the rational/structure-based drug design paradigmis based on the availability of several experimental crystal structuresof the target entity, namely the pathogen's protein synthesis machinery,embodied by the bacterial ribosome or subunits thereof, having a knownand pre-verified ligands thereof bound thereto. The paradigm includesthe identification and recordation of the pharmacophore associated witheach of the pre-verified ligands, and in particular the interaction offunctional groups in the active-site with moieties in each of thepre-verified ligands. According to some embodiments, the de-novo ligandpresented herein is designed as adduct (a conjugate) of two or moremolecular entities or moiety, each representing, mimicking orduplicating a moiety in a pre-existing antibacterial agent which hasbeen pre-verified by in-vitro and/or in-vivo bioactivity assays. In suchembodiments the possession of positioning data of the SA50S subunit(i.e., the molecular target) allows the design of the adduct such thatone moiety is attached to the other moiety so as to permit all moietiesto bind with its respective ribofunctional locus, preferablysimultaneously, thereby providing a highly effective andspecies-specific antibacterial agent that disrupts protein synthesis inthe target ribosomal subunit.

Non-limiting examples of pre-existing antibacterial agents includelinezolid, BC-3205 and telithromycin.

The resulting designed ligand, according to some embodiments of thepresent invention, has a molecular weight no greater than about 1,500grams/mol, no greater than about 1,000 grams/mol, no greater than 750grams/mol or no greater than about 500 grams/mol. The designed ligandpreferably has a molecular weight in the range from about 250 grams/molto about 1500 grams/mol, or in the range from about 500 grams/mol toabout 1200 grams/mol.

According to some embodiments, the affinity of the ligand to theribosomal subunit can be characterized by the association/dissociationconstant (interchangeably referred to herein as an affinity constant, abinding constant or k_(D)) of the ligand to the ribosomal subunit, asdetectable by a binding assay. According to some embodiment of thepresent invention the binding affinity is high and considered “specific”if it occurs with a k_(D) of 1 mM or less, generally in the range of 500μM to 10 pM.

According to some embodiments of the present invention, the designedligand has a minimal inhibitor concentration lower than 50 μM, lowerthan 10 μM, or lower than 1 μM, required for inhibiting 50 percentactivity (IC₅₀) in a biological assay, for example, an in vitrotranslation assay, for example, an SA translation assay. The designedligand has an IC₅₀ in the range of from about 0.001 μM to about 50 μM,or in the range of from about 0.01 μM to about 10 μM, or in the range offrom about 0.1 μM to about 1 μM.

In some of any of the respective embodiments of the invention, asubstantially rigid ligand is designed, such that the ligand haverelatively small number of conformations it can take by virtue of havinga relatively small number of rotatable bonds. It should be appreciatedthat absolutely rigid molecules are generally not feasible, hence theterm “substantially rigid” is used. Without being bound by anyparticular theory, it is assumed that as the structure of a givenmolecule is characterized by a higher degree of freedom and the lessrigid the molecule is, the harder it is for the molecule to bind to areceptor and the less tight is its binding. However, less rigidmolecules with some degree of conformational freedom are contemplated,taking into account design and/or synthetic and/or pharmaceuticalconsiderations, as well as other practical reasons.

In some of any of the respective embodiments of the present invention,the ligand is a pathogenic bacterium-specific protein synthesisinhibitor; in some of the embodiments, the ligand is a protein synthesisinhibitor of a Gram positive cocci bacterium; in some of theembodiments, the ligand is a protein synthesis inhibitor of aStaphylococcus bacterium; in some of the respective embodiments, theligand is a protein synthesis inhibitor of Streptococcus pneumoniae; insome of the respective embodiments, the ligand is a protein synthesisinhibitor of Bacillus subtilis; in some of the respective embodiments,the ligand is a protein synthesis inhibitor of Clostridium difficile;and in some of the respective embodiments, the ligand is a proteinsynthesis inhibitor of Staphylococcus aureus.

It is noted that the novel designed ligand for a ribosomal subunit of apathogenic microorganism is not a naturally occurring ligand, such as apeptide, a protein or an aminoglycoside, but it may exhibit somechemical elements (moieties) which are similar or mimic moieties of somenaturally occurring chemical entities or ligands. For example, while thenovel ligand is not a peptide, it made comprise one or more peptide(amide) binds, one or more amino acids bound by a peptide bond and thelikes, however the ligand comprises other chemical moieties that renderit clearly distinguishable from any naturally occurring peptide that mayserve as a naturally occurring ligand to the target ribosome.

A Method of Treating an Infection:

Treatment includes abscess drainage, debridement of necrotic tissue,removal of foreign bodies (including intravascular catheters), and useof antibacterial agents. Antibacterial agents are typically selectedbased on severity of the infection and local resistance patterns.Initial choice and dosage of antibiotics depend on infection site,illness severity, and probability that resistant strains are involved.Thus, it is useful to acquire information pertaining to local resistancepatterns for initial therapy, and ultimately, to monitor actual drugsusceptibility.

Treatment of toxin-medicated staphylococcal disease (disease caused byGram positive pathogenic bacterium such as Gram positive coccibacterium), most serious of which is toxic shock syndrome, involves:decontamination of the toxin-producing area by exploration of surgicalwounds, irrigation and debridement; intensive support by IV fluids,vasopressors and respiratory assistance; electrolyte balancing; and theuse of antimicrobial agents. In vitro evidence supports a preference forprotein synthesis inhibitors over other classes of antibiotics.

It is noted that many staphylococcal strains produce penicillinase, anenzyme that inactivates several β-lactam antibiotics; these strains areresistant to penicillin G, ampicillin and antipseudomonal penicillins.Community-acquired strains are often susceptible topenicillinase-resistant penicillins, such as methicillin, oxacillin,nafcillin, cloxacillin, dicloxacillin, cephalosporins, carbapenems,imipenem, meropenem, ertapenem, doripenem, macrolides, fluoroquinolones,trimethoprim/sulfamethoxazole (TMP/SMX), gentamicin, vancomycin andteicoplanin.

MRSA isolates have become common, especially in hospitals. Theseresistant strains, although resistant to most β-lactams, are usuallysusceptible to TMP/SMX, doxycycline, or minocycline and are oftensusceptible to clindamycin, but there is the potential for emergence ofclindamycin resistance by strains inducibly resistant to erythromycin.Vancomycin is effective against most MRSA, sometimes with rifampin andan aminoglycoside added for serious infections. Some alternative drugssuch as daptomycin, linezolid, quinupristin/dalfopristin, TMP-SMX andceftaroline, may be considered when treating MRSA strains with avancomycin.

Vancomycin-resistant S. aureus (VRSA) andvancomycin-intermediate-susceptible S. aureus (VISA) strains haveappeared in the North America. Control over these organisms requires theuse of linezolid, quinupristin/dalfopristin, daptomycin, TMP/SMX orceftaroline.

Because incidence of resistant SA strains has increased, initial empirictreatment for serious staphylococcal infections, particularly those thatoccur in a health care setting, typically include a drug with reliableactivity against MRSA. Thus, for proven or suspected bloodstreaminfections, vancomycin or daptomycin are typically used. For pneumonia,vancomycin or linezolid are used because daptomycin is not reliablyactive in the lungs.

It is noted that all of the above known methods of treatment are limitedin their efficacy due to growing rate of resistance immergence amongpathogenic strains of bacteria, and the presently provided ribosomal 50Ssubunit ligands offer a comprehensive solution to the problemsassociated with the treatments against such pathogenic strains by virtueof being a genus of rationally designed ligands that have been designedbased on the structural information afforded by the presently disclosedcrystal structure of the native and AB-complex SA50S exemplarystructures.

The resulting ligands can be useful in treating, inhibiting orpreventing the biological activities of target organisms, therebykilling the organism or impeding its growth. Alternatively, theresulting molecules can be useful for treating, inhibiting or preventingmicrobial, e.g., bacterial, infections in any organism, particularlyanimals, more particularly humans.

According to an aspect of some embodiments of the present invention,there is provided a method of treating an infection associated with apathogenic bacterium. The method is effected by administering to asubject in need thereof a therapeutically effective amount of the ligandpresented herein.

In some of any of the embodiments of the present invention, thepathogenic bacterium is a pathogenic Gram positive bacterium, or apathogenic bacterium exhibiting a degree of 23S rRNA sequence identityof at least 80% compared to the 23S rRNA of Staphylococcus aureus; insome of the respective embodiments, the pathogenic bacterium is a Grampositive cocci bacterium; in some of the respective embodiments, thepathogenic bacterium is a Staphylococcus bacterium; in some of therespective embodiments, the pathogenic bacterium is Streptococcuspneumoniae; in some of the respective embodiments, the pathogenicbacterium is Bacillus subtilis; in some of the respective embodiments,the pathogenic bacterium is Clostridium difficile; and in some of therespective embodiments, the pathogenic bacterium is Staphylococcusaureus.

According to some of any of the respective embodiments of the presentinvention, the pathogenic bacterium is a Gram positive cocci bacterium,a Staphylococcus bacterium or Staphylococcus aureus, and in some of theembodiments the Staphylococcus aureus is a drug-resistant strainthereof.

According to some embodiments, the ligand is administered alone or incombination with one or more other antibacterial agents.

According to some embodiments, the infection associated with apathogenic bacterium is a skin infection, which is one of the mostcommon types of disease produced by Staphylococcus bacterium (Staphinfections). Staph infections of the skin can progress to impetigo (acrusting of the skin) or cellulitis (inflammation of the deeper layersof skin and connective tissue under the skin, leading to swelling andredness of the area). In some cases, a serious complication known asscalded skin syndrome can develop from Staph infections. Inbreastfeeding women, Staph infections can result in mastitis(inflammation of the breast) or in abscess of the breast. Staphylococcalbreast abscesses can release bacteria into the mother's milk.

When the Staphylococcus bacteria enter the bloodstream and spread toother organs, a number of infections can occur. Spread of the organismsto the bloodstream is known as bacteremia or sepsis. Staphylococcalpneumonia predominantly affects people with underlying lung disease andcan lead to abscess formation within the lungs. Infection of the heartvalves (endocarditis) can lead to heart failure. Spread of Staphylococcibacteria to the bones can result in severe inflammation of the bonesknown as osteomyelitis. When Staphylococci bacteria are present in theblood, a condition known as staphylococcal sepsis (widespread infectionof the bloodstream) or staphylococcal bacteremia exists. Staphylococcalsepsis is a leading cause of shock and circulatory collapse, leading todeath, in people with severe burns over large areas of the body. Whenuntreated, S. aureus sepsis carries a mortality rate of over 80%. S.aureus has been reported as a cause of chorioamnionitis and neonatalsepsis in pregnancy, leading to life-threatening condition for thefetus.

Staphylococcal infections are contagious and can be transmitted fromperson to person.

A Pharmaceutical Composition:

According to an aspect of some embodiments of the present invention,there is provided a pharmaceutical composition which includes as anactive ingredient, the ligand of the ribosomal subunit of a pathogenicbacterium, as provided herein.

In some of any of the respective embodiments of the present invention,the pharmaceutical composition is packaged in a packaging material andidentified in print, in or on the packaging material, for use in thetreatment of an infection associated with a pathogenic bacterium, e.g.,S. aureus infection.

The active ligand, once identified according to some embodiments of thepresent invention, may be incorporated into any suitable carrier priorto use. More specifically, the dose of active ligand, mode ofadministration and use of suitable carrier will depend upon the targetand non-target organism of interest.

With regard to mammalian recipients, the active ligand may beadministered by any conventional approach known and/or used in the art.Thus, as appropriate, administration can be oral or parenteral,including intravenous and intraperitoneal routes of administration. Inaddition, administration can be by periodic injections of a bolus, orcan be made more continuous by intravenous or intraperitonealadministration from a reservoir which is external (e.g. an intravenousbag). In certain embodiments, the active ligand of the invention can betherapeutic-grade, namely certain embodiments comply with standards ofpurity and quality control required for administration to humans.Veterinary applications are also within the intended meaning as usedherein.

The formulations, both for veterinary and for human medical use, of theactive ligand according to the present embodiments, typically includesuch agents in association with a pharmaceutically acceptable carrier,and optionally other therapeutic ingredient(s). The carrier(s) should be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.Pharmaceutically acceptable carriers, in this regard, are intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ligand, use thereof in the compositions is contemplated.Supplementary active agents, identified or designed according to theinvention and/or known in the art, also can be incorporated into thecompositions. The formulations may conveniently be presented in dosageunit form and may be prepared by any of the methods well known in theart of pharmacology/microbiology. In general, some formulations areprepared by bringing the active ligand into association with a liquidcarrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation.

Pharmaceutical compositions according to some embodiments of the presentinvention are formulated to be compatible with its intended route ofadministration. Examples of routes of administration include oral orparenteral, e.g., intravenous, intradermal, inhalation, transdermal(topical), transmucosal, and rectal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide.

Useful solutions for oral or parenteral administration can be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., (1990).

Pharmaceutical compositions suitable for injectable use, according tosome embodiments of the present invention, include sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor ELTM (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS).

The active ligand may be prepared with carriers that will protect theligand against rapid elimination from the body, such as a controlledrelease formulation, including implants and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialsalso can be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. Microsomes and microparticles also can be used.

As noted above, active ligands identified or designed according toembodiments of the present invention can be formulated intopharmaceutical compositions by admixture with pharmaceuticallyacceptable nontoxic excipients and carriers. Such compositions can beprepared for parenteral administration, particularly in the form ofliquid solutions or suspensions; for oral administration, particularlyin the form of tablets or capsules; or intranasally, particularly in theform of powders, nasal drops or aerosols. Where adhesion to a tissuesurface is desired the composition can include the active liganddispersed in a fibrinogen-thrombin composition or other bioadhesive. Theactive ligand can then be painted, sprayed or otherwise applied to thedesired tissue surface. Alternatively, the active ligand can beformulated for parenteral or oral administration to humans or othermammals, for example, in therapeutically effective amounts, e.g.,amounts which provide appropriate concentrations of the active ligand totarget tissue for a time sufficient to induce the desired effect.

Active ligands identified or designed by any of the methods providedherein may also come in a form of precursors of the active ligands. Theterm “precursor” refers to a pharmacologically inactive (or partiallyinactive) derivative of a parent ligand that requires biotransformation,either spontaneous or enzymatic, within the organism to release theactive ligands. Precursors are variations or derivatives of the ligandsof the invention which have groups cleavable under metabolic conditions.Precursors become the active ligands of the invention which arepharmaceutically active in vivo, when they undergo solvolysis underphysiological conditions or undergo enzymatic degradation. Precursorforms often offer advantages of solubility, tissue compatibility, ordelayed release in the mammalian organism (see Bundgard, Design ofProdrugs, pp. 7-9, 21-24, Elsevier, Amsterdam (1985); and Silverman, TheOrganic Chemistry of Drug Design and Drug Action, pp. 352-401, AcademicPress, San Diego, Calif. (1992).

Active ligands as identified or designed by any of the methods describedherein can be administered to individuals to treat disorders(prophylactically or therapeutically). In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a drug as well as tailoringthe dosage and/or therapeutic regimen of treatment with the drug.

With regard to mammals, it is contemplated that the effective dose of aligand that serves as a protein synthesis inducer or inhibitor, will bein the range of about 0.01 to about 50 mg/kg, preferably about 0.1 toabout 10 mg/kg of body weight, administered in single or multiple doses.Typically, the ligand may be administered to a human recipient in needof treatment at a daily dose range of about 1 to about 2000 mg perpatient.

It is expected that during the life of a patent maturing from thisapplication additional relevant structures of large ribosomal subunit ofpathogenic bacteria and SA50S-specific ligands will be developed and thescope of the terms SA50S structures and SA50S-specific ligands isintended to include all such new technologies a priori.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

When reference is made to particular sequence listings, such referenceis to be understood to also encompass sequences that substantiallycorrespond to its complementary sequence as including minor sequencevariations, resulting from, e.g., sequencing errors, cloning errors, orother alterations resulting in base substitution, base deletion or baseaddition, provided that the frequency of such variations is less than 1in 50 nucleotides, alternatively, less than 1 in 100 nucleotides,alternatively, less than 1 in 200 nucleotides, alternatively, less than1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides,alternatively, less than 1 in 5,000 nucleotides, alternatively, lessthan 1 in 10,000 nucleotides.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Example 1 Preparation of 50S Subunit Sample from SA

SA Growth and Cell Wall Disruption:

Following Iordanescu and Surdeanu [Journal of General Microbiology,1976, 96(2), p. 277-81] Staphylococcus aureus (SA) strain RN4220 (ATCC35556) was grown overnight at 37° C., and cells were harvested atOD_(600nm) of about 1.5. The bacterial culture was centrifuged twice ina table top centrifuge for 10 minutes at 4000 rpm at 4° C. Thesupernatants were discarded and the wet cell pellets were weighed,resuspended in 10 mM Tris-Acetate buffer, pH=8.0, 14 mM Mg-Acetate, 1 MKCl, 1 mM DTT and 50 μg/ml lysostaphin (glycylglycine endopeptidase thatbreaks down the cell wall Staphylococci species), incubated at 37° C.for 1 hour and periodically inverted.

The lysates were centrifuged for 30 minutes at 36,000 rpm, at 4° C. forremoving cell debris. The supernatants were incubated in 670 mMTris-Acetate buffer pH=8.0, 20 mM Mg-Acetate, 7 mM DTT, 7 mMNa₃-phosphoenolpyruvate, 5.5 mM ATP, 70 mM from each amino acid and 1.9mg Pyruvatkinase at 37° C. for 30 minutes and dialyzed over night at 4°C. against dialysis 10 mM Tris-Acetate buffer pH=8.0, 14 mM Mg-Acetate,60 mM K-Acetate and 1 mM DTT.

The extract was then flash-frozen and stored at −80° C.

Purification of SA Ribosomes:

Cell extract was layered on a sucrose cushion (Selmer et al., 2006) 1.1M sucrose, 10 mM Hepes (pH=8.0, pH was set according to the pH of thecell extract), 15 mM MgCl₂, 100 mM NH₄Cl, 50 mM KCl, 6 mMβ-mercaptoethanol, and ultracentrifuged twice, each time for 17 hrs at4° C. at 55 Krpm using a Ti-70 rotor. The supernatant was thendiscarded; the pellet was dissolved in 10 mM Hepes, 15 mM MgCl₂, 150 mMNH₄Cl, 50 mM KCl and 6 mM β-mercaptoethanol buffer set at pH=8.0.Ribosomal subunits were then separated by zonal ultracentrifugation,using a Ti-15 zonal rotor with a gradient of 8-40% sucrose, keeping lowMg²⁺ concentration (1 mM MgCl₂) for 17.5 hours at 27K rpm. Afterseparation, the Mg²⁺ concentration was increased to 10 mM and theribosomal subunits fractions were collected and concentrated usingsequential centrifugations. The samples were kept at 10 mM Hepes buffer,10 mM MgCl₂, 60 mM NH₄Cl and 15 mM KCl buffer set at pH=7.6, and broughtto a final concentration of 600-1000 or 800-1000 A₂₆₀ ml⁻¹, then wereflash-frozen for storage at −80° C.

Ribosome Activity Assay:

The SA ribosomes activity was tested and its level was determined in abacterial coupled transcription/translation assay system which measuresthe expression of the luciferase gene, according to Murray, R. W. et al.[Antimicrobial agents and chemotherapy, 2001, 45(6), p. 1900-1904].Briefly, the luciferase gene was inserted into plasmid with T7 RNApolymerase promoter. The 30 μl reaction's volume contained 160 mMHepes-KOH buffer (pH 7.5), 6.5% PEG 8K, 0.074 mg/ml tyrosine, 1.3 mMATP, 0.86 mM CTP, GTP and UTP, 208 mM potassium glutamate, 83 mMcreatine phosphate, 28 mM NH₄OAc, 0.663 mM cAMP, 1.8 mM DTT, 0.036 mg/mlfolinic acid, 0.174 mg/ml E. coli tRNA mix, 1 mM amino acid, 0.25 mg/mlcreatine kinase, 0.027 mg/ml T7 RNA polymerase, ribosome free E. colicell free extract, 300 nM of SA ribosomes and 0.003 μg/μl luciferaseplasmid.

The reaction mixture was incubated at 37° C. for 1 hour, anderythromycin was added at a final concentration of 8 μM in order toterminate the reaction. 50 μl of Luciferin Assay Reagent (LAR, Promega)was added to the mixture and luminescence was measured in order toquantify the reaction's products.

Example 2 Crystallization of 50S Subunit from SA

Native SA50S Crystals:

Crystals of the 50S ribosomal subunit extracted from Staphylococcusaureus (SA50S) were obtained at 20° C. by the hanging-drop vapordiffusion technique. The crystallization solution contained 0.166%2-methyl-2,4-pentanediol (MPD), 0.333% EtOH, 20 mM Hepes, 10 mM MgCl₂,60 mM NH₄Cl and 15 mM KCl buffer set to pH range of 6.8-7.8, 5 mMspermidine, 0.5 mM MnCl₂ and 1-1.6 mg/ml SA50S. The reservoir solutioncontained 15% of 1:2 ethanol-MPD and 110 mM Hepes, 10 mM MgCl₂, 60 mMNH₄Cl and 15 mM KCl buffer. The SA50S subunits were heat activated for30 minutes at 37° C. before crystallization. These conditions usuallyyield about 60-300 μm hexagonal crystals, which appeared as hexagons.High resolution diffracting crystals were obtained by macro seeding,using crystals that were extracted from the crystallization drop, washedin 10 μl of 7.5% EtOH, 7.5% MPD, 110 mM Hepes, 10 mM MgCl₂, 60 mM NH₄Cland 15 mM KCl buffer and 0.5 mM MnCl₂, and seeded in a crystallizationdrop as described hereinabove, pre-equilibrated for 24 hours.

SA50S crystals were kept in a stabilization solution containing 15% MPD,15% EtOH, 110 mM Hepes, 10 mM MgCl₂, 60 mM NH₄Cl and 15 mM KCl bufferset to pH range of 6.8-7.8) and 0.5 mM MnCl₂.

Crystals of SA50S Complexes:

For obtaining SA50S antibiotics complexes, SA50S crystals obtained asdescribed hereinabove were soaked in solutions containing 6 μg/mllinezolid, 11.4-22.7 μg/ml BC-3205, or 16.2 μg/ml telithromycin in thestabilization solution for 3-6 hours prior to exposure to X-ray and datacollection.

Example 3 Crystal Structure of 50s Subunit from SA

Data Collection and Processing:

Prior to exposure to X-ray, the crystals were immersed in acryoprotectant solution containing 20% MPD, 15% EtOH, 110 mM Hepes, 10mM MgCl₂, 60 mM NH₄Cl and 15 mM KCl buffer and 0.5 mM MnCl₂.Crystallographic X-ray diffraction data were collected at the ID23-1,ID23-2 and ID-29 beamlines, at the European Synchrotron RadiationFacility (ESRF), Grenoble, France, from the hexagonal crystals at atemperature of 100° K. Up to 15 SA50S crystals were used for yielding acomplete dataset using 0.1 degree oscillations. Data were processed withHKL-2000 [Otwinowski, Z. and Minor, W., Methods in Enzymology,Macromolecular Crystallography, part A, 1997, 276, p. 307-326, Carter,C. W. Jr. and Sweet, R. M., Eds., Academic Press (New York)] and CCP4package suite [Winn, M. D. et al., Acta Crystallographica Section D:Biological Crystallography, 2011, 67, p. 235-242].

Electron Density Map Calculation, Model Building and Refinement:

The structures were determined by molecular replacement using PHASERimplemented in PHENIX [McCoy, A. J. et al., J Appl Crystallogr, 2007,40, p. 658-674], using the structure of D50S (PDB ID: 2ZJR) as astarting model. Once initial phases were obtained, rigid body andpositional refinement were performed using Phenix.refine [Afonine, P. V.et al., Acta Crystallogr D Biol Crystallogr, 2012, 68, p. 352-67] andCNS [Brunger A. T. et al., Acta Crystallogr D Biol Crystallogr, 1998,54(5), p. 905-21].

For R-free calculations during refinement cycles, random 5% of the datawere omitted during refinement. Tracing the ribosomal RNA and remodelingthe ribosomal proteins with SA strain NCTC8325 sequence according to theelectron density maps was performed using Coot [Emsley, P. et al., ActaCrystallogr D Biol Crystallogr., 2010, 66(4), p. 486-501], RosettaERRASER [Chou, F. C. et al., Nat Methods., 2013, 10(1), p. 74-6] wasused to facilitate further building and to improve the quality of theRNA geometry. Figures were generated using Pymol [The PyMOL MolecularGraphics System, Version 1.5.0.4 Schrödinger, LLC]. Sequence alignmentswere performed using BLAST [Altschul, S. F. et al., J Mol Biol., 1990,215(3), p. 403-10] and presented by JalView [Waterhouse, A. M. et al.,Bioinformatics, 2009, 25, p. 1189-1191]. Structure alignments wereperformed using LSQMAN [Kleywegt, G. J. and Jones, T. A., Structure,1995, 3, p. 535-540] and Coot.

FIG. 1 presents a graphic illustration of the large ribosomal subunit ofSA (SA50S), wherein the rRNA is colored in grey, the rProteins arecolored in various colors, the PTC is marked by a red star and theapproximate path of the internal exit tunnel is marked by a band coloredin dark blue.

In summary, the structures of the large ribosomal subunit fromStaphylococcus aureus and of its complexes with linezolid, telithromycinand BC-3205 were determined by X-ray crystallography, and the data wererefined using molecular replacement phasing. Table 3 presents a summaryof the crystallographic data and structure refinement statistics ofnative SA50S structure (SA50S), SA50S structure in complex withlinezolid (SA50Slin), SA50S structure in complex with BC-3205(SA50SBC-3205) and SA50S structure in complex with telithromycin(SA50Steli). Ninety three percent of the nucleotides and most of theamino acid residues of 26 of SA50S rProteins (ribosomal proteins) weretraced in the electron density map. Also detected was electron densityputatively assigned to hydrated ions such as Mg²⁺ and Mn²⁺.

TABLE 3 Subject SA50S SA50Slin SA50SBC-3205 SA50Steli Crystalinformation Space group P6₅22 P6₅22 P6₅22 P6₅22 a [Å] 279.6 279.9 280.9282.7 b [Å] 279.6 279.9 280.9 282.7 c [Å] 872.7 870.6 875.6 877.1 α, β,γ [°] 90, 90, 120 90, 90, 120 90, 90, 120 90, 90, 120 Complex with —linezolid BC-3205 telithromycin Diffraction data statistics X-ray sourceID23-1/2 (ESRF) ID23-1 (ESRF) ID23-2 (ESRF) ID23-1 (ESRF) Wavelength [Å]0.873, 0.972 and 1.00 0.972 and 1.00 0.873 0.973 Number of crystals 11 820 11 Crystal oscillation [°] 0.1 0.1 0.1 0.1 Resolution [Å]   50-3.51(3.57-3.51)  200-3.4 (3.46-3.4)   50-3.43 (3.47-3.43)   50-3.45(3.51-3.45) Unique reflections 236855 246474 253918 257382 Observedreflections 2813593 1392748 2109298 1792405 Redundancy 12 (6)  5.7 (2.8)8.3 (3.4) 7 (5) Completeness [%] 96.3 (84.3) 89.4 (64.8) 92.1 (78.9)98.5 (96.5) <I>/<σ>  7.4 (1.43) 8.45 (1.16) 10.51 (1.21)  8.84 (1.21)R-merge [%] 25.3 (97.8) 15.6 (77.6) 15.2 (74.4) 16.5 (92.6) RefinementR-factor [%] 20.18 20.87 20.42 19.53 R-free (5%) [%] 24.65 25.23 24.223.57 RMSD bonds [Å] 0.006 0.006 0.006 0.006 RMSD angles [°] 1.161 1.121.099 1.118

FIGS. 2A-F present the weighted 2F_(o)-F_(c) electron density maps oflinezolid (FIG. 2A), telithromycin (FIG. 2B) and BC-3205 (FIG. 2C),contoured at 1.0 σ, and the weighted F_(o)-F_(c) electron density mapsof linezolid (FIG. 2D), telithromycin (FIG. 2E) and BC-3205 (FIG. 2F),contoured at 3.0 σ.

Example 4 Main Cross-Species Structural Variability

Structural Variability in rRNA Between Species:

Sequence alignment of the 23S rRNA shows 81%, 76% and 73% identitybetween S. aureus and D. radiodurans, E. coli, and T. thermophilus,respectively, suggesting almost identical rRNA structures with somediversity. Since this diversity may highlight clues for speciesspecificity in resistant mechanisms stemming from the unique features ofSA50S structure, its structure was compared with the structures of D50S(PDB ID: 2ZJR), T70S (PDB IDs: 2WDL and 2WDK) and E70S (PDB IDs: 2AW4and 3R8S).

The crystal structure comparison revealed significant similarities inthe rRNA folds of all high resolution structures available to date.Nevertheless, several internal structural differences, alongside someregions that are located on the surface of the 50S subunit wereidentified, which seem to originate from sequence variability ratherthan from crystal packing interactions. Some of such regions are locatedmainly at the subunit interface within the active ribosome. The generallocations of the variable rRNA regions within the entire large subunitare shown in FIGS. 3A-B and close-up illustrations for each region areshown in FIGS. 4A-H.

FIGS. 3A-B present a graphic illustration of the structure of SA50Sshowing relative locations of the rRNA regions with fold variability onthe SA50S subunit, wherein SA50S 23S rRNA is shown in teal, the variableregions are shown in orange and (FIG. 3A and FIG. 3B) are rotated 90°with respect to each other.

FIGS. 4A-H present graphic illustrations of superimposed structuremodels of S. aureus (colored in teal), D. radiodurans (colored in grey),E. coli (colored in purple) and T. thermophilus (colored in orange),showing the structural variability in the rRNA backbone, wherein (FIG.4A) emphasizes the h25 region, (FIG. 4B) emphasizes the h9 region, (FIG.4C) emphasizes the h63 region, (FIG. 4D) emphasizes the h10 region,(FIG. 4E) emphasizes the h79 region, (FIG. 4F) emphasizes the h15 andh16 regions, (FIG. 4G) emphasizes the h68 region, and (FIG. 4H)emphasizes the h28 region.

Herein and throughout, lower case “h” is used as a prefix for thenumbers of the rRNA helices.

As can be seen in FIGS. 4A-H, a rather significant fold variance wasobserved in the h28 region; in SA50S it possesses a differentorientation compared to the other three structures (FIG. 4H). Additionalexamples are found in the h25 and h9 regions that have different foldsin all 4 structures (FIGS. 4A and B); the h63 region, which is inproximity to the intersubunit bridge B5 and has a different length ineach of the four structures, longest is in E70S, shorter in SA50S andT70S and the shortest in D50S (FIG. 4C). Helices h10 and h79, which areinteracting with each other and with rProteins L28 and L2, respectively,are extended in SA50S and E70S compared to their conformation in T70Sand D50S (FIGS. 4D and E). The h16 region of SA50S and E70S is thelonger compared to that of T70S and D50S (FIG. 4F). Interestingly, h15does not exist in E70S crystal structure and could not be traced inD50S, whereas it is clearly resolved and shows small structuraldiversity between SA50S and T70S, indicating a varying level offlexibility (FIG. 4F).

As can further be seen in FIGS. 4A-H, the h68 region, which is involvedin the binding of rProtein L1, EF-P (elongation factor-G), RRF(ribosomal recycling factor) and belongs to the intersubunit bridge B7awith the 30S subunit, is longer in SA50S than the h68 of T70S and D50S;however it is not fully traced in SA50S structure (FIG. 4G).

FIGS. 5A-B present graphic illustrations of the flexible nucleotides atthe PTC and at the exit tunnel (FIG. 5A), showing U2506, U2585, A2062,A2602 and U2491, where the P-site tRNA (shown as a green surface) andthe A-site tRNA (shown as a blue surface) would bind, whereas S. aureus23S RNA backbone and nucleotides are colored in teal, and D.radiodurans, T. thermophilus and E. coli nucleotides are shown in grey,orange and purple, respectively, and further showing the flexiblenucleotides towards the tunnel opening (FIG. 5B), wherein A90, A91 andA508 are located in the ribosomal exit tunnel, detected with differentconformations in all 4 structures, and a possible path of the backboneof a modeled nascent poly-alanine chain is represented by a yellowstring.

As can be seen in FIGS. 5A-B, within the core of the ribosome, the rRNAfold is mostly conserved in the four eubacterial ribosome structures,including the previously identified flexible nucleotides with variableconformations at the PTC, namely U2506, U2585, A2062, A2602, U2491 andat the exit tunnel, namely A508 and A90-91.

Structural Variability in rProteins Between Species:

Sequence alignment of the large ribosomal subunit proteins showed anoverall identity of about 50% between the S. aureus, E. coli, T.thermophilus and D. radiodurans.

The crystal structure comparison of the rProteins showed a greaterdiversity in their detailed structures compared to the differencesobserved for the rRNA structure. Typically, the main features of therProteins folds, such as the globular domains and the secondarystructure elements that interact with the rRNA are rather conserved;however, several SA50S rProteins contain extensions that were not seenin any other ribosome structure previously published. Relative positionsof the globular domains of the rProteins on the subunit surface areshown in FIGS. 6A-D.

FIGS. 6A-D present a graphic illustrations of the surface of the SA50Sindicating the locations of the globular regions of the rProteins,whereas rRNA is shown in grey and the various rProteins are shown indifferent colors, showing a view from the SA50S intersubunit surface(FIG. 6A), a view from the SA50S outer surface (FIG. 6B), and views of a+90 degrees and −90 degrees vertical rotation of the intersubunitsurface (FIGS. 6C and D respectively).

It is noted that almost all the rProteins contain N- and/or C-terminalextensions, as well as extended internal loops (both types may reach70-80 Å in length), which penetrate into the ribosome core and interactwith the ribosomal RNA. These extensions contain most of the structuralvariability of the rProteins.

SA rProteins numbering is used throughout the comparative analysis owingto the significant variability in the rProteins sequences and folds ofthe four compared structures.

The Inter-Subunit Interface:

FIGS. 7A-B present a graphic illustrations of the subunit interface,showing some of the structural differences of the rProteins, whilefocusing on L5 (FIG. 7A) and L16 (FIG. 7B).

As can be seen in FIGS. 7A-B, there is a notable structural variabilityin M38-A53 and G126-D144 loops of L5 that participates in the B1bintersubunit bridge with 30S subunit (16S, colored in grey) withrProtein S13 (colored in dark green) (FIG. 7A). Also the SA50S L16 isextended compared to T70S, E70S and D50S structures thus may form aunique interaction net with A-site tRNA acceptor stem (colored in blue)and the P-site tRNA (colored in green) interacts with the loop L77-V90that has some structural variability (FIG. 7B). By superposition of theSA50S subunit on the corresponding part in T70S, one can identify thestructural variability in M38-A53 and G126-D144 loops of L5 thatinteract with rProtein S13 in the 70S ribosome, embracing S13 interfacehelix in all four structures (FIG. 7A). The L16 protein is positioned inthe intersubunit surface and interacts with the A-site and P-site tRNAs,superposed on PDB ID: 2WDK. The N-terminal of L16 SA50S is extendedcompared to T70S, E70S and D50S structures thus may form a uniqueinteraction network with A-site tRNA acceptor stem (see, FIG. 7B).

Furthermore, the fold of the N-terminal of protein L2 in SA50S issomewhat different from that of E70S, in which L2 is longer.Interestingly in D50S, 30 residues of protein L2 could not be traced,whereas in SA50S only 14 residues could be traced. The rProtein L5participates in the B1 intersubunit bridge with the 30S subunit,together with the rProtein S13.

FIGS. 8A-B present a graphic illustration that emphasizes the structuraldifferences between SA50S, T70S, E70S and D50S in the rProteins thatinteract with substrates, while focusing on L28 (FIG. 8A) and L27 (FIG.8B).

As can be seen in FIG. 8A, protein L28 is located close the 50S surface,where the CCA 5′ of the E-site tRNA (shown in yellow) binds, and the L28in D50S and T70S has a 15 residues extended loop (S19-K27) that reachesh11 of the 23S rRNA. This loop is shorter in SA50S and E70S thatinteracts with h21 and h75. As can be seen in FIG. 8B, L27 in D50Sreaches to the acceptor stem of the P-site tRNA (shown in green),whereas in T70S it reaches the PTC, in the proximity of the CCA end ofthe P-site tRNA. Additional differences found in L14 structures arewithin the core of the subunit may facilitate substrate stabilizationduring protein synthesis. As previously observed, the flexibleN-terminal of L27 appears in several conformations in four differentlyobtained crystal structures. In the presently provided SA50S crystalstructure, the N-terminal of L27 is discernible by in the electrondensity map (can be traced) from residue 19 (based on sequencealignment) and is folded slightly differently than in the other 50Sstructures from other species. It has been previously reported that SAhas an extended L27 N-terminus which is being cleavedpost-translationally by a specific and essential protease, prior to orconcurrent with ribosome assembly. Therefore in the presently providedSA50S crystal structure only 10 residues of the N-terminal are notmodeled (traced) due to a fragmented electron density, confirming theflexibility of this part of the structure. In the E70S crystal structurea shift of 4-5 residues is suggested, based on sequence alignment of L27on T. thermophilus, D. radiodurans and SA, and thus indicating that only4-5 amino acid residues cannot be traced in the electron density map dueto local chain flexibility and motility. In D50S, where almost theentire N-terminal was traced in the electron density map, and no tRNAexists in the crystal, superposition on the T70S with 3 tRNA moleculesin the complex, indicating that it may reach the acceptor stem of theP-site tRNA, whereas in T70S it reaches the PTC interacting with the CCA3′end of the P-site tRNA (see, FIG. 8B).

The Nascent Protein Exit Tunnel:

The exit tunnel is of around a 100 Å in length. It has a non-uniformshape with a variable diameter. It contains a constriction located about5-7 peptide bonds away from its entrance, which is shaped at its farend, by the hairpin loops of rProteins L4 and L22 that reach the tunnelwalls in all four compared structures despite some small sequence andstructural variability.

L4 loop of S. aureus is more hydrophobic compared to the other comparedbacterial species. From the opposite tunnel wall, the L22 hydrophobichairpin loop has a similar fold in all structures compared; however, theorientations of R92 (R/S/R in T70S, D50S and E70S) are somewhatdifferent in all four structures. It is noted that the macrolide (e.g.,Erythromycin) binding site is proximal to this constriction.

FIGS. 9A-G present graphic illustrations that emphasizes the structuraldifferences between SA50S, T70S, E70S and D50S in the rProteins at thewalls of the nascent protein exit tunnel, the subunit's core and centralprotuberance, whereas the possible path of the backbone of a modelednascent protein chain is indicated in lime/yellow in some of theillustrations.

As can be seen in FIGS. 9A-G, L32 N-terminal in D50S and T70S iselongated relative to SA50S and E70S, thus reached the rims of theerythromycin binding site (FIG. 9A); a view into the exit tunnel openingshows structural variability of the rProteins L23, L24, and L29 (FIG.9B); the L24 is compared in all 4 structures (FIG. 9C). The L18N-terminal domain is elongated in SA relative to D50S, T70S and E70Sreaching the other side of the central protuberance (FIG. 9D); the L25domains are shown, as well as structural variability in the fold ofG11-L26, I49-T69, F79-186 loops, and the L16 C-terminal is longer inT70S than in SA50S, D50S and E70S (FIG. 9E); a zoom-into view of thehinge between the L25 domains shows that the L16 C-terminal is longer inT70S structure than in SA50S, D50S and E70S where it penetrates into theproximity of L25, thus changing the angle between L25 domains (FIG. 9F);and the L27 R79-K85 loop and C-terminal fold exhibits variability amongthe 4 structures (FIG. 9G).

The N-terminals of L32 in D50S and T70S are elongated relative to SA50Sand E70S, thus creating a void proximal to the macrolides binding site.This void may be exploited for potential new or improved drugs (i.e.extended macrolides). In addition, L32 V24-Q37 that reaches the subunitsurface, possess structural variability among the 4 structures. ItsC-terminal is 14-17 residues shorter in SA50S than in D50S, T70S andE70S.

At the vicinity of the tunnel opening structural variations wereobserved in ribosomal proteins L23, L29, L24, L32 and L22, creatingdifferent surfaces for ribosomes interactions with cellular factors,such as the translocon, ER membranes, chaperons and the likes. The L23K63-Y71 loop that is pointing into the tunnel, displays a differentconformation in SA50S and T70S in comparison to E70S and D50S.Specifically, in the four compared structures, residue R67 (Q in E70S)points into the tunnel in different orientations. The tyrosine in D50Sstacks to A508 nucleotide but A508 has a different conformation in eachof the 4 structures, which underlines its flexibility (see, FIG. 4B).The L23 C-terminal of SA50S is similar in length to its mates in D50Sand T70S, but its fold is different from that of E70S, which has anextended C-terminal tail. Consequently, this region of the subunitsurface that interacts with other cell components as the ER membrane orchaperons is altered among the four structures examined (see, FIG. 4B).

The L24 N-terminal interacts with h7 and the junction between h18, h19and h20 of the 23S rRNA. The D50S N-terminal is 10-13 residues longerthan in the other 3 structures. The SA50S and T70S N-terminals areshorter (the SA50S N-terminal first two amino acids could not be traceddue to not interpretable density) and the E70S N-terminal is theshortest. The E70S L24 I24-V34 loop folds different than in SA50S, D50Sand T70S whereas K43-G55 loop of SA50S folds similar to T70S exhibitingextended loop, different from E70S and D50S. Additionally, the 5′ end ofL24 T79-I93 loop and the C-terminal tail are highly divergent among the4 structures (FIG. 9C). In E70S, the L37-T40 loop of rProtein L29 isextended compared to the 3 other structures (FIG. 9B).

Subunit Core and the Central Protuberance:

The rProteins globular regions are located on the surface of the largeribosome subunit. Their long extended tails and loops penetrate into thesubunit core and interact with the rRNA. Since differences in proteinsthat are interacting with the rRNA may result in allosteric alterationsin the nucleotides, several substantial structural variability inribosomal core proteins are noted herein.

FIGS. 10A-H present graphic illustrations emphasizing the structuraldifferences between S. aureus (colored in teal), D. radiodurans (coloredin grey), T. thermophilus (colored in orange) and E. coli (colored inpurple) in some rProteins at the subunit surface.

As can be seen in FIGS. 10A-H, SA50S rProtein L3 has a unique extendedA57-L67 loop compared to those of each of D50S, T70S and E70S (FIG.10A), and that the SA50S L17 has an extended (T65-A81) loop which isunique to SA50S, its C-terminal is about 10 amino-acids longer than inE70S, and it is also longer than its counterparts in the other 3organisms (FIG. 10B). The length variations detected in the V6-I17surface loop of protein L4 and N-terminal of L15 that is located in itsvicinity, whereas the most extended is found in SA50S, less extended inE70S and D50S and has altogether different orientation in T70S (FIG.10C). The C and N-terminals of L4 has different backbone folds in thefour eubacterial structures (FIGS. 10D-E). SA50S L15 loops I69-T89 andT89-V97 have different structures compared to the corresponding loops inD50S, T70S and E70S (FIG. 10F). The L15 N-terminal in E70S, D50S (tracedfrom the 4th amino acid), T70S (traced from the 5th amino acid), is theshortest in SA50S (traced from the 1st amino acid) (FIG. 10G). The T70SL28 globular domain has a fold that differ significantly from all otherL28 structures compared (FIG. 10H).

L14 loop L25-S28 is extended in D50S compared to SA50S, E70S and T70Sstructures and makes a unique interaction with h95 in addition to thecommon interaction with h90. L15 N-terminal is a long extended strandwith different fold among the compared structures (SA50S exhibiting isthe shortest L15 N-terminal) that penetrates into the subunits core(see, FIGS. 10C and 10G), and interacting with protein L35 which has asimilar fold in the four compared structures. The N-terminal of SA50SL17, which is located in proximity to h100-h101, h61 and h96, is 4residues shorter relative to other 3 structures compared (FIG. 10B).

The central protuberance which lies on the surface of the 50S subunit isa sub-complex of the 5S rRNA with rProteins L5, L18, L25, L27 and L25(referred to as TL5 in the context of T50S and CTC in the context ofD50S). This rProtein interacts with the 5S rRNA. In E70S rProtein L25 isbuilt of a single domain, in SA50S and T70S it has two domains and inD50S the CTC protein contains three domains. The first L25 domain ofSA50S and E70S has an extended G11-L26 loop that interacts with 5S rRNA.The F79-I86 loops that interact with the 23S rRNA have differentbackbone folds and orientations in the four compared structures. TheC-terminal of L16 R134-T141 reaches the L25 domains interface and 5SrRNA. The N-terminal of L16 is longer in T70S and penetrating deeperinto the domains interface, thus, the angle between the two TL5 domainsis larger compared to D50S and SA50S (see, FIGS. 9D and 9E). L18N-terminal in SA50S is longer than in E70S, D50S and T70S. ItsN-terminal has been traced only from the eighth and eleventh amino acidrespectively. It reaches rProtein L5 that is located on the other sideof the 5S rRNA (see, FIG. 9D). The rProtein L27 has a similar structurein all four compared structures apart from variability in the fold ofR79-K85 loop that reaches the 5S rRNA in all structures. Structuralvariations of the C-terminal that reaches the subunit surface are alsoobserved (see, FIG. 9F) in addition to the gross differences in theN-terminal tail mentioned above.

The Subunit Surface:

Among the structural differences identified in the structures of thelarge ribosomal subunits that are located on their surfaces is SA50SrProtein L3 that has an extended (H58 R69) loop next to h100 of 23S rRNA(see, FIG. 10A). Similarly, rProtein L17 of SA50S has a unique extendedT65-A81 loop; its C-terminal is about 10 amino acid residues longer thanin E70S, and is also longer than in the other three compared structures.In both L3 and L17 these differences cause a different shape of thesubunit surface (see, FIG. 10B). Also, the N- and C-terminals of L29,which reach the subunit surface, possess variable folds in the fourcompared structures. The N-terminals are extended in T70S and E70Scompared to their folds in SA50S and D50S structures, whereas theC-terminals are of similar length but their fold is variable.

In addition, variability in SA50S L3 backbone fold compared with D50S,T70S and E70S was observed. Among them E29-V34 and F91-A112 and loopsV15-P24, S142-P151, G152-P170 and Q187-K198 have a different fold andorientation in SA50S compared to T70S and E70S (see, FIG. 10A and FIG.13A). The L4 of SA50S has an extended V6-I17 surface loop compared tothis loop in E70S and D50S, which in T70S adopts a different orientation(see, FIG. 10C). In addition, in SA50S L4 N-terminal has a differentbackbone fold, Q119-E132 (see, FIG. 10E), and different structure andorientation of its C-terminal tail (I186-T195) (FIG. 10D) compared toE70S, T70S and D50S.

The SA50S L15 C-terminal domain is located on the surface of the 50Ssubunit. Its loops I69-T89 and T89-V97 have different structure comparedto the corresponding loops of E70S, but similar to T70S and D50SC-terminal domain (see, FIG. 10F). FIG. 10C shows the surface shared byL4 and L15 and their overall structural differences among the fourdifferent crystal structures. The L15 N-terminal is the longest in E70S,shorter in D50S (traced from the fourth amino acid residue) and shorterin T70S (traced from the fifth amino acid residue). The L15 N-terminalis the shortest in SA50S (traced from the first amino acid residue) butits 70 amino acids residues extended the N-terminal tail intercalateddifferently with the 23S rRNA of the 4 structures compared (see, FIGS.10F and 10G).

Furthermore, although L19 N-terminus of SA50S and D50S are positioneddifferently, both interact with h101 while D50S L19 N-terminus interactswith h100 and L3 and while T70S and E70S N-terminals are shorter. InSA50S L21 N-terminal, K24-D31 loop and G43-V58 loop have a slightlydifferent backbone fold than the other three compared structures. TheN-terminal of D50S L22 is 22-residues longer compared to SA50S, T70S andE70S resulting in a rather different surface of the D50S subunit. Inaddition, the core structure of L28, which is situated in proximity tothe outer surface of the large subunit, is folded differently in thefour compared structures. Also, the L28 I38-W48 loop adopts a differentorientation in SA50S compared to E70S, T70S and D50S (FIG. 10H).

The L30 N-terminal that is located on the particle's outer surface ispositioned differently in SA50S and T50S structures compared to E70S,whereas the D50S N-terminal is shorter. The L30 C-terminal, that alsoreaches the subunit outer surface, is longer in T70S compared to theother three compared structures.

FIGS. 11A-B present graphic illustrations emphasizing the structuraldifferences between SA50S, T70S, E70S and D50S in the N-terminal ofprotein L32 that resides in the second shell around the erythromycinbinding pocket.

As can be seen in FIG. 11A, the L32 is shorter in SA50S (colored inteal) as well as in E70S (colored in purple) compared to its length inthe ribosomes of the non-pathogenic bacteria D. radiodurans (colored ingrey) and T. thermophilus (colored in orange) as seen in FIG. 11B.

In summary, some of the differences in rProteins detected in SA include:

(i) the length of the N-terminus of rProtein L17 (see, FIG. 10B);

(ii) the flexible N-terminal of rProtein L27 that displays structuralfold variability in proximity to the acceptor stem and 3′ end of P-sitetRNA (see, FIG. 8B);

(iii) a shorter loop in rProtein L28 that lies in proximity to E-sitetRNA; and

(iv) a significantly shorter N-terminal region of rProtein L32 thatresults in the creation of an additional void at the rims of themacrolides binding site.

Thus, in D50S and T70S the N-terminal tails of L32 reach theerythromycin binding pocket, whereas in the pathogenic SA50S and thepotential pathogenic E70S they are distal to this pocket (see, FIGS. 11Aand 11B). Notably, in most bacteria L32 N-terminal is shorter, similarto what is seen for SA50S and for E70S (see seq. alignment attached). Inaddition, in all known structures of eubacterial ribosomes nucleotideA2058 is stacked onto G2057 that interacts with residue 5 of L32N-terminal tail. The sequence and structure variability of this residue(Lys in SA50S, T70S and D50S and Gln in E70S), creates differentenvironments around A2057.

A detailed comparative structural analysis presenting the mainstructural differences in SA50S rProteins and rRNA is summarized inTable 4 and Table 5 respectively.

TABLE 4 rProtein Residues Difference Ribosome Site FIG. L2 N-terminalVariation in its length and fold. 30 residues are intersubunit surface,SEQ ID NO. 3 missing from the D50S structure. In the current next to h34SA50S structure it is traced from the 3^(rd) residue. L3 V15-P24Different fold and orientation compared with subunit surface next 10ASEQ ID NO. 4 T70S and E70S. to L14 and L19 L3 E29-V34 Variability inbackbone folds in all four subunit surface 10A SEQ ID NO. 4 structures.within its globular domain L3 A57-L67 SA50S extended loop next to h100of 23S rRNA, subunit surface next 10A SEQ ID NO. 4 compared to E70S,D50S and T70S. to h100 L3 F91-A112 Variability in backbone folds in allfour subunit surface 10A SEQ ID NO. 4 structures. within its globulardomain L3 H134-P170 Different fold and orientation compared with subunitcore, in 13B SEQ ID NO. 4 T70S and E70S. between h61, h35 and h90 L3Q187-K198 Different fold and orientation compared with subunit surface10A SEQ ID NO. 4 T70S and E70S. within its globular domain L4 N-terminalDifferent backbone folds in the four structures. subunit surface 10E SEQID NO. 5 within its globular domain, next to h29 L4 V6-I17 Extendedsurface loop compared to this loop in subunit surface 10C SEQ ID NO. 5E70S and D50S. This loop adopts a different within its globularorientation in T70S structure. domain L4 W65 W65 of SA50S (W/W/Y inE70S, T70S and Protein exit tunnel 13E, 13F SEQ ID NO. 5 D50S) pointsinto the exit tunnel and has a entrance different orientation relativeto the other structures. L4 K66 K66 in SA50S (R/G/P in E70S, D50S andT70S) Protein exit tunnel 13E, 13F SEQ ID NO. 5 interacts withnucleotide U790 in the 23S rRNA entrance that is pointing into the exittunnel. L4 K68 Conserved among all 4 structures. Situated in Proteinexit tunnel 13E, 13F SEQ ID NO. 5 proximity to the PTC nucleotides A2060and entrance U2061, but exhibits a different orientation in each of the4 structures. L4 R72 R72 in SA50S (R/R/N in E70S, T70S and D50S) Proteinexit tunnel 13E, 13F SEQ ID NO. 5 points into the exit tunnel indifferent orientations entrance in the four structures. L4 Q119-E132Different backbone fold in the four structures subunit surface, 10E SEQID NO. 5 close to h19, h20 junction L4 C-terminal Different structureand orientation in the four subunit surface 10E SEQ ID NO. 5 structures.within its globular domain, next to h28 L5 M38-A53 High structuralvariability observed among four part of intersubunit  7A SEQ ID NO. 6structures. bridge B1b L5 G126-D144 High structural variability observedamong four part of intersubunit  7A SEQ ID NO. 6 structures. bridge B1bL14 L25-S28 Extended loop in D50S compared to SA50S, Subunit core in SEQID NO. 9 E70S and T70S, forming a unique interaction between h90, h92with h95 in addition to the common interaction and h95 with h90. L15N-terminal L15 N-terminal is a long extended strand with reaches subunit10C SEQ ID NO. 10 fold variation among the four structures that surface,next to L4 begins at the subunit surface and penetrates into the core ofthe 23S rRNA. In SA50S this loop is the shortest. L15 I69-V97 Differentstructure of this loop in the four subunit surface 10F SEQ ID NO. 10structures. within its globular domain, close to h28 L16 N-terminalSA50S L16 N-terminal is extended compared to intersubunit surface,  7BSEQ ID NO. 11 T70S, E70S and D50S structures thus may form between h38and unique interactions with A-site tRNA acceptor h89, A-site tRNA stem.binding site. L16 and L25 C-terminal T70S C-terminal is longer,penetrating deeper central protuberance,  9F SEQ ID NOs. between L25domains thus, the angle between the subunit surface 11 and 20 L25domains is larger compared to D50S and SA50S L25 structures. L17N-terminal SA50S N-terminal is four residues shorter Subunit core in 10BSEQ ID NO. 12 relative to other 3 structures compared and it betweenh96, h91 folds differently. and h100 L17 T65-A81 This loop is mostextended in SA50S. subunit surface 10B SEQ ID NO. 12 between h47, h59and h57 L17 C-terminal ~10aa longer in E70S compared to other threesubunit surface 10B SEQ ID NO. 12 structures, thus is extended at thesubunit surface. within its globular domain, close to h101 L18N-terminal SA50S N-terminal is longer than E70S tail, centralprotuberance,  9D SEQ ID NO. 13 reaching protein L5 across the 5S rRNA.In D50S subunit surface and T70S this N-terminal has been traced onlyfrom aa 8 and 11 respectively. L18 T53-A73 Variable fold among the fourstructures. central protuberance,  9D SEQ ID NO. 13 subunit surface L19N-terminal SA50S L19 N-terminus interacts with h101 subunit surface nextSEQ ID NO. 14 whereas D50S L19 N-terminus interacts with to h101 h100and L3. T70S and E70S N-terminus are shorter. L21 N-terminal SA50Sbackbone fold is slightly different than subunit surface SEQ ID NO. 16the backbone fold in the other three structures. within its globulardomain next to L20 L21 K24-D31 SA50S backbone fold is slightly differentthan subunit surface next SEQ ID NO. 16 the backbone fold in the otherthree structures. to h45 L21 G43-V58 SA50S backbone fold is slightlydifferent than subunit surface SEQ ID NO. 16 the backbone fold in theother 3 structures. within its globular domain next to L20 L22N-terminal D50S N-terminal is 22 residues longer compared subunitsurface next SEQ ID NO. 17 to SA50S, T70S and E70S resulting in a ratherto h24 different surface of the D50S subunit at this face. L22 K83 ThisLys side chain in SA interacts with Protein exit tunnel SEQ ID NO. 17nucleotide C1261 (h26) adopting a different entrance orientationcompared with E70S, T70S and D50S structures. L22 R84 R84 in SA50S, E70Sand T70S points in different Protein exit tunnel SEQ ID NO. 17 directionthan in D50S. entrance L22 S85-F94 Hydrophobic loop that foldsdifferently in Protein exit tunnel SEQ ID NO. 17 SA50S, D50 and T70Scompared to E70S entrance L22 R86 Only in SA50S residue R86 (I/L/M inD50S, Protein exit tunnel SEQ ID NO. 17 T70S and E70S) reachesnucleotide C1325 (h50) entrance U/A/A in E70S, D50S and T70S. L22 Q90 Qin SA and R in D50S and T70S. Pointing into Protein exit tunnel SEQ IDNO. 17 the exit tunnel. Glutamine side chain is shorter entrance thanarginine, allowing a wider passage in the tunnel. Since E. coli has adifferent backbone fold in this region, the aligned residue is K andit's in a different position. L22 R92 R/S/R in T70S, D50S and E70S.Orientation is Protein exit tunnel SEQ ID NO. 17 different among allfour structures. entrance L23 K63-Y71 Pointing into the tunnel, displaysa 3 different Protein exit tunnel  4B SEQ ID NO. 18 orientation betweenSA50S/T70S, D50S and E70S . . . Especially, residue R67 (Q in E70S)which points into the tunnel in different directions L23 C-terminalSA50S C-terminal tail is similar in length to Protein exit tunnel SEQ IDNO. 18 D50S and T70S. It folds different from E70S that end (surface)exhibits extended C-terminal tail. Consequently this part of the subunitsurface, which interacts with the ER membrane, is altered among the fourstructures. L24 N-terminal Reaches the outer subunit surface viainteractions subunit surface via  9C SEQ ID NO. 19 with h7 and thejunction h18, h19, h20 of the 23S interactions with the rRNA. In D50Sthe N-terminal of L24 is 10-13 h7 and the junction residues longer thanin the other 3 structures. The h18, h19, h20 SA50S and T70S N-terminalsare shorter (the SA50S N-terminal is partly not traced due touninterruptable density). The E70S N-terminal is the shortest. L24I24-V34 The E70S loop folds different than in SA50S, surface next to h7 9C SEQ ID NO. 19 D50S and T70S. L24 K43-G55 This loop has a differentorientation and a slightly Protein exit tunnel  9C SEQ ID NO. 19different fold between all four structures. end (surface) next to h24and h7 L24 T79-I93 Highly divergent among the four structures. surfaceof the  9C SEQ ID NO. 19 subunit between L7 and L18 L24 C-terminalHighly divergent among the four structures. surface, close to h7 SEQ IDNO. 19 L25 domains L25 of E70S is a single domain protein whereascentral protuberance,  9E SEQ ID NO. 20 in SA50S and T70S L25 iscomposed of 2 subunit surface domains and in D50S L25 is composed of 3domains. L25 G11-L26 SA50S and E70S have an extended loop that centralprotuberance,  9E SEQ ID NO. 20 interacts with 5S rRNA. subunit surfaceL25 I49-T69 Variable backbone fold and orientation in all four centralprotuberance, SEQ ID NO. 20 structures. subunit surface L25 F79-I86Variable backbone fold and orientation in all four central protuberance,SEQ ID NO. 20 structures. subunit surface L27 N-terminal SA50S L27 istruncated as could be tracing only intersubunit surface  8B SEQ ID NO.21 from aa 19 (by sequence alignment). Its positioning is somewhatdifferent compared to T70S (complex with tRNA) and D50S embracing thesuperimposed P-site tRNA. In E70S (2AW4) structure N-terminal tail isthe shortest and also embracing the superimposed P-site tRNA. L27R79-K85 Variable fold among the four structures. central protuberance, 9G SEQ ID NO. 21 subunit surface L27 C-terminal Structural variations,as reported earlier for all central protuberance,  9G SEQ ID NO. 21known structures (Maguire, B. A 2005). subunit surface L28 Entireprotein The T70S L28 has different fold compared with intersubunitsurface 10H SEQ ID NO. 22 its fold in E70S, D50S and SA50S structures.L28 S19-K27 In D50S and T70S L28 has an extended loop, intersubunitsurface,  8A SEQ ID NO. 22 compared to E70S and SA50S, that reaches h11between h11 and h21 of the 23S rRNA and should interact with the CCA 5′of E-site tRNA. L28 I38-W48 Adopts a different orientation in SA50Sintersubunit surface 10H SEQ ID NO. 22 compared to E70S, T70S and D50Sbetween h79 and h10 L29 N-terminal Extended in T70S and E70S compared toSA50S subunit surface, next SEQ ID NO. 23 and D50S N-terminalstructures. to h7 L29 L37- T40 E70S loop is more extended than in the 3other Protein exit tunnel  9B SEQ ID NO. 23 structures. exit (surface)L29 C-terminal Variable folds among the four structures. subunit surfacenext SEQ ID NO. 23 to h18 L30 N-terminal Pointing in differentdirections in SA50S and subunit surface close SEQ ID NO. 24 T50Sstructures compared to E70S. In D50S the to its C-terminal andN-terminal is shorter. h38 L30 C-terminal Longer in T70S compared to theother three subunit surface close SEQ ID NO. 24 structures. to itsN-terminal and h38 L32 N- terminal D50S and T70S are elongated relativeto SA50S Protein exit tunnel 9A, 11A, SEQ ID NO. 25 and E70S, thuspenetrating deeper into the tunnel entrance 11B wall. L32 V24-Q37Structural variability among the four structures surface between L22 SEQID NO. 25 globular domain h101 and h99

TABLE 5 23S rRNA SEQ ID NO. 1 Difference Ribosome Site FIG. h9 Differentfold among the four structures compared. Surface, in vicinity to 4B L23h10 Elongated in SA50S and E70S structures relative to T70S interactingwith h79 4D and D50S structures. and with proteins L28 h15-h16 In SA50Sh15 and h16 fold differently than in E70S and Surface, in vicinity to 4Fh16 in D50S. Interestingly, h15 is missing in the E70S L28 structure andcould not be traced in D50S, presumably owing to its flexibility. h25Different fold among the four structures compared. Surface, in vicinityto 4A L21 h28 Different fold among the four structures compared. next tothe surface of 4H rProtein L4 globular domain h63 Variable lengths inall four structures, longest in E70S, in proximity to 4C shorter inSA50S and T70S and shortest in D50S. intersubunit bridge B5 h68 Notfully traced in SA50S structure as it is longer than h68 involved in the4G of T70S and D50S structures. binding of rProtein L1, EF-P, RRF and ispart of the intersubunit bridge B7a h79 Elongated in SA50S and E70Srelative to T70S and D50S. interacting with h10 4E and with proteins L2U2506, U2504, Flexible nucleotides. PTC 5A U2609, U2585, A2062, A2602(h93), U2491 (h89) A508, A90- Flexible nucleotides. exit tunnel exit, on5B A91 (h7) the surface of the subunit

Example 5 The Complex Crystal Structure of SA50S with AntibacterialAgents

FIGS. 12A-F present graphic illustrations of linezolid bound to SA50S(SA50S in complex with linezolid), referred to herein as SA50Slin (A andB), telithromycin bound to SA50S, referred to herein as SA50Steli (C andD) and BC-3205 bound to SA50S, referred to herein as SA50SBC-3205 (E andF).

FIG. 12A shows a comparison of native SA50S peptidyl transferase center(PTC) structure (colored in teal) and SA50Slin complex (colored in paleorange). Hydrogen bonds between linezolid (colored in orange) and 23SrRNA are shown in black dashes. FIG. 12B shows an overlay of thestructures of various ribosome-linezolid complexes [SA50Slin (colored inpale orange), H50Slin (colored in green) complexed also with CCA-Phesubstrate analog (colored in teal) (PDB ID: 3PCW), D50Slin (colored ingrey) (PDB ID: 3DLL) and of the model of E70Slin (colored in pink). Thecolor code of the rRNA components of the various linezolid bindingpockets is the same as of corresponding linezolid molecules. FIG. 12Cshows a comparison between native SA50S PTC (colored in teal) andSA50Steli complex (colored in red). The main hydrogen bond betweentelithromycin (colored in slate) and 23S rRNA is shown in black dashes.FIG. 12D shows a structural overlay of various telithromycinconformations observed in various ribosome-telithromycin complexstructures, presenting SA50Steli (colored in slate), D50Steli (coloredin orange) (PDB ID: 1P9X), H50Steli (colored in grey) (PDB ID: 1YIJ),E70Steli (colored in pink) (PDB ID: 3OAT), and T70Steli (colored ingreen) (PDB ID: 3OI3). The color code of the rRNA components of thevarious telithromycin binding pockets is in brighter tone than thecorresponding telithromycin molecules. FIG. 12E shows a comparisonbetween the structure of the PTC in native SA50S (colored in teal) andin SA50SBC-3205 (colored in purple), the arrows in this figure show themovements of nucleotides U2585 and U2506 in the bound versus the nativestructure. Hydrogen bonds between BC-3205 (colored in violet) and 23SrRNA are shown as black dashes. FIG. 12F shows a structural overlay ofvarious pleuromutilins in their binding pockets in (colored in violet),D50S-SB571519 (colored in green) (PDB ID: 2OGM), D50S-retapamulin(colored in cyan) (PDB ID: 2OGO), D50S-tiamulin (colored in slate) (PDBID: 1XBP), and D50S-SB280080 (colored in lemon) (PDB ID: 2OGN). Only onehydrogen bond between BC-3205 (colored in violet) and 23S rRNA is shownas black dashes.

Crystal Structure of SA50S-Linezolid Complex:

The SA50S-linezolid complex structure (SA50Slin) revealed that linezolidis bound at the PTC, blocking the A-site, thus perturbing tRNAaccommodation, in an orientation grossly similar to that observed inother ribosome linezolid complexes with D50S, H50S and the E50Slinmodel. However, in SA50Slin complex the flexible nucleotide U2585undergoes a significant rotation and forms a hydrogen bond with the 04of the linezolid morpholino ring, which yields a nonproductiveconformation of the PTC (see, FIGS. 12A and 12B).

The flexibility of U2585 plays a role in the rotatory motion of thetranslocation of the tRNA 3′ end from the A-site to the P-site. Hence,fixing its conformation by a hydrogen bond with linezolid shouldparalyze the PTC catalytic activity. The linezolid 1,3-oxazolidin-2-onemoiety and acetamide group form additional hydrogen bonds with G2505 andA2451, respectively. The fluorophenyl moiety of linezolid is located ina heteroaromatic crevice formed by the PTC residues C2451 and C2452, theso-called A-site cleft. All of its other interactions with the rRNAnucleotides, namely G2061, C2501, U2504, U2506 and G2447, are either Vander Waals or hydrophobic interactions.

Comparing SA50Slin structure (see, FIG. 12A) with other availablecrystal structures or models of large ribosomal subunits in complex withlinezolid, namely H50Slin, D50Slin and E70Slin model (see, FIG. 12B)revealed that in all structures the drug is bound at the same pocket;however, there are subtle differences between the conformation of thelinezolid acetamide group in SA50Slin and the other structures. Theseinclude a 100-120 degree rotation of the acetomide group which, inSA50Slin enables fixation of A2451 by a hydrogen bond. In addition, inH50Slin complex, the NH of the acetomide group is forming a hydrogenbond with the oxygen on the phosphate of G2505, whereas in D50Slincomplex no such specific interaction is observed. Noteworthy, H50Slincomplex was crystallized in the presence of CCA-Phe (tRNA 3′ endsubstrate analogue), assuming that in such environment it shouldinteracts strongly with the 50S subunit P-site. Indeed, it seems thatthis P-site analog altered linezolid conformation, compared to otherlinezolid complex structures that were determined with empty P-site.Interestingly, a hydrogen bond between the morpholino ring of linezolidand nucleotide U2585 was observed in D50Slin and in SA50Slin, but not inH50Slin. Additionally, for detecting linezolid in the H50Slin crystalstructure the drug concentration was three orders of magnitude higherthan the concentrations used for linezolid soaking into D50S or SA50Scrystals.

The Structure of SA50S-Telithromycin Complex:

The SA50Steli structure shows that telithromycin is bound at theMLS_(B)K binding site, and forms the typical ketolides (and macrolides)hydrogen bond between its desosamine sugar and A2058. At this positionit is partly blocking the protein exit tunnel, as was found in otherribosome-MLS_(B)K complex structures [Schlünzen, F. et al., MolecularMicrobiology, 2004, 54(5), p. 1287-1294; Belousoff, M. J. et al.,P.N.A.S USA, 2011, 108(7), p. 2717-2722 and Berisio, R. et al., JBacteriol., 2003, 185(14), p. 4276-9]. In SA50Steli complex the flexiblenucleotide U2062 is rotated compared to its conformation in native SA50Sand its conformational range is minimized by the hydrogen bond withA2503 (see, FIG. 12C). All of its other interactions with the rRNAnucleotides, namely G2505, A2059, C2611 and U746, are either hydrophobicinteractions or based on Van der Waals distances.

Comparing SA50Steli structure with other available crystal structures ofribosomal particles in complex with telithromycin [Berisio, R. et al., JBacteriol., 2003, 185(14), p. 4276-9; Tu, D et al., Cell, 2005, 121(2),p. 257-70; Dunkle, J. A. et al., Proc Natl Acad Sci USA, 2010, 107(40),p. 17152-17157; and Bulkley, D. et al., Proc Natl Acad Sci USA, 2010,107(40), p. 17158-63] revealed that in all structures the drug is boundat the same pocket but with distinct differences in the orientations ofthe alkyl-aryl moiety. Similar orientation of this moiety was seen inSA50Steli and in H50Steli. In both, the arm is folded back over themacrolactone ring, thus creating a rather compact structure of the drug;however, in SA50Steli the alkyl-aryl arm is reaching the center of thetunnel about 5 Å closer to the PTC compared to its location in H50Steliand may block the nascent protein progression in an earlier stage. Inthis orientation the alkyl-aryl arm is almost overlapping the locationof the cladinose sugar of erythromycin in its complex with D50S (see,FIG. 13A). In contrast, in T70Steli and E70Steli this arm is stacked toA752 and U2609, a base pair that is located on the tunnel wall furtheraway from the PTC, so that it can block nascent protein progressionabout 10 Å away from the point of blockage observed in H50Steli. InD50Steli, the alkyl-aryl arm is extended, thus blocks the tunnel byinteracting with U790 across the tunnel, creating a barrier located 15 Åfurther along the tunnel compared to SA50Steli (see, FIG. 12D). It hasbeen suggested that the structure of E70Steli reflects the telithromycinbinding mode to the ribosomes of medically relevant (namely pathogenic)eubacteria species, since A752-U2690 base pair is conserved among alleubacteria [Dunkle, J. A. et al., Proc Natl Acad Sci USA, 2010, 107(40),p. 17152-17157], and in addition, telithromycin resistant Streptococcuspneumonia AA752 mutant has been isolated and characterized.Nevertheless, in SA50Steli the alkyl-aryl arm of telithromycin does notinteract with A752-U2690 base-pair, thus demonstrating that antibiotic(antibacterial agent) binding modes are species specific; hence, clearlydemonstrating that general description of the overall antibioticsbinding properties is the main outcome from the previous structurescannot be extrapolated safely to other species.

The Structure of SA50S-BC3205 Complex:

In SA50SBC-3205 complex structure, BC-3205 is bound at the PTC, wherethe tricyclic mutilin core is blocking the A-site, and its C14 extensionis pointing into the P-site, thus perturbing A-site and P-site tRNAaccommodation, as was found in other ribosome-pleuromutilin complexstructures with D50S. In SA50SBC-3205 the conformation of the flexiblenucleotide U2585 is different from that of the unbound SA50S and itsconformational range is reduced because of partial overlap by theBC-3205 (see, FIG. 12E). In addition, U2506 is shifted towards the wallsof the binding pocket, forming a hydrogen bond between its 04 carbonylto the valyl moiety NH₂ of BC-3205. An additional hydrogen bond isformed between the acetyl carbonyl and the NH₂ of G2061. All of itsother interactions with the rRNA nucleotides, namely A2063, A2503,U2504, G2505, A2451, C2452 and U2585, are either hydrophobicinteractions or based on Van der Waals distances.

Comparing SA50SBC-3205 with the crystal structures of D50S in complexwith various pleuromutilins (see, FIG. 12F) revealed that allpleuromutilins bind to the same pocket, albeit by somewhat differentinteractions, including a shift of U2585. In SA50SBC-3205, this shift islarger than in all other pleuromutilin complexes so that the base ofU2585 is located about 6 Å away from its position in the nativeconformation. Similarly, a movement of U2506 towards the bound drug wasobserved in pleuromutilins complexes with D50S. Notably, inSA50SBC-3205, U2506 forms a hydrogen bond with the NH₂ of the valylmoiety of the drug, hence its shift is the largest. Consequently the twosides of BC-3205 are held within its binding pocket by hydrogen bonds,compared to the single hydrogen bond created in the other pleuromutilinscomplexes with G2061, thus indicating a better fit of BC-3205 to itsbinding site. This additional interaction of BC-3205 seems to accountfor its higher potency against MRSA resistant strains and its low IC₅₀value.

Example 6 Structural Analysis of Known SA Resistance Mechanisms

FIGS. 13A-F present graphic illustrations of structures of native SA50SrRNA and rProteins (colored in teal), D50S (colored in grey), E70S(colored in purple) and T70S (colored in orange) superimposed forcomparative analysis and study of the resistance and cross resistancemechanisms in SA, showing rRNA nucleotides of SA in regions where theycan be well aligned with the corresponding nucleotides in all otherstructures used for the comparisons.

FIG. 13A shows SA telithromycin (colored in slate) conformation withinits SA complex where its alkyl-aryl arm is folded such that it overlapsthe desosamine sugar of erythromycin (colored in red) in its complexwith D50S. SA50teli L22 (colored in blue) is superimposed on rProteininsertion resistant mutant of L22 (colored in red) that allows nascentprotein progression although it binds erythromycin (PDB ID: 4U67). FIG.13B shows SA50S rRNA nucleotides of the linezolid (colored in orange)and BC-3205 (colored in green) binding sites are superimposed on thecorresponding E70S rRNA (colored in purple). G2576 is located in the 2ndshell around the linezolid and BC-3205 binding sites, in proximity tothe 1st shell nucleotides G2505 and U2506 thus G2576 mutation may causealterations in them. The locations of the L3 mutations acquiringresistant of SA are marked on the protein chain (colored in yellow). Forcomparison, the structure of E70SL3 (colored in purple) is superposed onSA50S L3 (colored in teal). Key structural differences are marked byarrows. The orange stars indicate deletions (SA numbering system isused). FIGS. 13C-D show linezolid (colored in orange), chloramphenicol(colored in green) and dalfoprostin (streptograminsA, colored in red) inthe SA50S rRNA binding pocket. FIG. 13E shows SA50S rRNA A2058 and A2059are main binding determinants of MLS_(B)K family of antibiotics,represented here by erythromycin (PDB ID: 3OFR) (colored in red).rProteins L4 and L32 form a second shell around erythromycin bindingpocket next to A5058 and A2059. Structure variability of L4 among D50S,E70S, T70S and SA50S is also shown. FIG. 13F shows rProtein L4 is invicinity to SA linezolid (colored in orange) and SA BC-3205 (colored ingreen) binding pockets. The structural variability of its loop (W65-Q75)in all fours species is also shown.

SA linezolid resistance is caused mainly by G2576U (SA G2603U) mutation,a nucleotide that is more than 98% conserved throughout all kingdoms,and located in the second shell around linezolid binding pocket, at thePTC, in vicinity to rProtein L3. This nucleotide is stacked to G2505that is located adjacent to U2504 and U2506. These three nucleotides arepart of the first shell around linezolid binding pocket and make directcontacts with linezolid in all available crystal structures of itscomplex with the large ribosomal subunit. It is conceivable that amutation in G2576 may cause alterations in these first shell nucleotidessince it is stacked to the first shell nucleotide G2505 and forms ahydrogen bond with U2506. Compared to E70S, the L3 loop G152-P170 inSA50S is bent towards the minor groove of h72 away from G2576, hencepermitting more flexibility (see, FIGS. 13B-C).

SA linezolid resistance is also caused by rRNA mutations in U2500A,A2503G, U2504C and G2447U nucleotides, which are more than 98% conservedthroughout all kingdoms. Within the SA50S, D50S and H50S-linezolidcomplexes, the drug interacts with U2504; mutating it to C will abolishthe CU base-pair interactions with C2452, thus may change the shape ofthe linezolid binding pocket. G2447 interacts with A2451 that is part ofthe linezolid binding pocket. The mutation G2447U may form a newbase-pair with A2451 that may limit the A2451 flexibility thusinfluencing drug binding. U2500 is base-paired with A2453, and itsmutation to A will abolish the base-pair interactions and consequentlychange the PTC environment (see, FIG. 13D). These nucleotides are partof a network that stabilizes the PTC, which is also the binding site ofa few antibiotics (antibacterial agents) from different families. Oncemutated, the interactions described above are eliminated and antibioticbinding does not occur. The G2447U mutation, which renders also M.smegmatis and M. tuberculosis linezolid resistant, confers lethality inE. coli and has been suggested to belong to the functional differencesbetween the ribosomes of Gram positive and Gram negative bacteria.Interestingly, significant structural similarity was observed in thisregion of ribosome from T70S, D50S, E70S and SA50S, which represent bothGram positive and Gram negative species. Such high cross-types (Grampositive and Gram negative) sequence similarity opens the path fordesigning ligands that are characterized by high affinity to largeribosomal subunits of both Gram positive and Gram negative bacteria.

Cross resistance to linezolid, chloramphenicol and dalfoprostin(streptograminA; a constituent of Synercid which contains alsostreptogramin B) is associated with the second shell nucleotide G2576Umutation. Linezolid, chloramphenicol and streptograminsA bind at the PTCvia the first shell nucleotide G2505 that is stacked to U2576. Inaddition, cross resistance of SA to linezolid and chloramphenicol, ofwhich the binding pockets partially overlap, may be caused by G2505A andU2500A mutations although the conservation level of these nucleotides ismore than 98% throughout all kingdoms. These mutations indicate thatdespite its high conservation, this nucleotide is not essential forribosomal function. Nucleotide G2505 is base-paired to C2610 and itsribose and phosphate are located in the vicinity of the antibiotic. Themutation G2505A leads to a mismatch in this base pair, thus altering theantibiotic binding pocket. Similarly, U2500 is base paired with A2453and has hydrophobic interactions with U2504. The mutation U2500A isdisrupting the base pair U2500-A2453, thus may enable U2504 tilting awayfrom the linezolid binding pocket (see, FIG. 13C).

SA resistant mutations are also associated with the region of rProteinL3 that is located in proximity of the PTC. This rProtein is involved inseveral resistance mechanisms, such as: (a) G152D and G155R that acquireresistance to linezolid, tiamulin, chlorampenicol and retapamulin, (b)ΔS145 acquire resistance to linezolid and tiamulin, (c) R1495 triggerresistance to tiamulin, (d) ΔF127-H146 acquire linezolid resistance, (e)S158L and D159Y cause resistance to tiamulin and retapamulin, (f) G144Dand S153Y acquire resistance to retapamulin and (g) the combinations ofG152D, D159Y, G144R or G152D, D159Y, G155R, H134N or G152D, D159Y,G155R, A150T acquire resistance to retapamulin, tiamulin andchloramphenicol. In SA50S, the fold of L3 loop G139-A150 is similar toits folds in D50S and T70S, all bending toward h90 and h62 (lower case“h” is used throughout as prefix for the numbers of the rRNA helices),but is different in E70S as it bends towards h61 major groove (see, FIG.13B). All mutated amino acids that were found in clinical resistantisolates are located in the L3 region that is adjacent to the thirdshell of nucleotides around the PTC, appear to reshape the antibioticsbinding pocket (colored in yellow in FIG. 13B). Mutations in the L3loops that are in vicinity of G2578 and C2575, which are part of thesecond shell nucleotides, may affect G2576 and its interactions withnucleotides G2505-U2506 that are part of the tiamulin and linezolidbinding pockets.

L3 loop F127-P170 is located also in the vicinity to G2574 thatinteracts with A2572, which is proximal to the flexible nucleotideU2504. In D50S and SA50S, this nucleotide is located in the first shellaround linezolid and tiamulin binding pockets, and interacts with them(see, FIG. 13B). This nucleotide has been previously implicated in PTCantibiotics resistance acquired by induced fit alterations that althoughnot directly interacting with the bound drugs, reshape the bindingpocket via networks of remote interactions, most of which through theflexible nucleotide U2504. In another SA linezolid resistance strain, adeletion of F127-H146 occurs in rplC gene, which is encoding rProteinL3. In L3 wild type, this loop is located within the area describedabove, in vicinity to 23S rRNA helix h90 nucleotides G2576-G2580, whichreside in the second shell around linezolid binding pocket. Loopdeletion may eliminate or alter interactions of L3 with its proximalrRNA, thus may reshape the drug binding pocket and hamper theantibiotics binding (see, FIG. 13B). Similarly, mutations in locationssomewhat distal from the actual antibiotic binding pockets that conferresistance by allosteric rearrangements have been detected in othersystems.

U2506 seems to be one of the most flexible nucleotides within thelinezolid and BC-3205 binding pockets. It adopts a differentconformation in each of the available crystal structures (see, FIG. 12B)[Wilson, D. N. et al., P.N.A.S. USA, 2008, 105(36), p. 13339-44;Ippolito, J. A. et al., J Med Chem, 2008, 51(12), p. 3353-6; Selmer, M.et al., Science, 2006, 313(5795), p. 1935-1942; and Schuwirth, B. S. etal., Science, 2005, 310(5749), p. 827-834], resulting in a significantlydifferent shapes of the linezolid binding pockets in the pathogen SAversus the archaeal H50S and the whole E70S ribosomes.

Nucleotides A2058 and A2059 are more than 98% conserved in eubacteria.Notably, in all higher organisms, including archaea, position 2058 is aguanine. These nucleotides, particularly A2058, are known as the maindeterminants for binding MLS_(B)K antibiotics by direct interactions(see, FIG. 13E) [Schlünzen, F. et al., Nature, 2001, 413(6858), p.814-821; Harms, Jorg M. et al., BMC biology, 2004, 2, p. 4]. Indeed, theidentity of the nucleotide in position 2058 plays a key role not only inpatient-pathogen discrimination, but also in resistance to MLS_(B)Kantibiotic families that is acquired either by A2058G mutation, or bypost posttranscriptional modifications performed by the erm genes, whichencode methyltransferase, an enzyme that methylates A2058 in SA as wellas in other eubacteria. Other mutations in this region, i.e. A2058U,A2058G and A2059G cause resistance to azithromycin and erythromycin inSA.

A2058 and A2059 are in direct contact with the W65-K68 loop of rProteinL4, which has diverse sequences in SA50S, E70S, T70S and D50S (see,FIGS. 13E-F). Mutations in rProtein L4, G69A and T70P acquire SAresistance to linezolid, Q67K, G69E, G69A and T70P mutations acquire SAresistance to erythromycin. In addition, AK68 and AG69 are susceptibleto linezolid and K68Q mutation acquires SA resistance to linezolid,pleuromutilins, chloramphenicol and tedezolid. Since L4 Q67-T70 loop islocated in the vicinity of the phosphate of A2059, which belongs to theMLS_(B)K binding pocket, the above mentioned mutations may alter theW65-Q75 loop conformation thus affecting A2059 conformation (see, FIGS.13E-F). In E70S, T70S and SA50S structures, residue R72 of L4 isarginine, whereas in D50S it is asparagine. Each of these four sidechains of residue 72 points to a different direction, creating differentenvironment around nucleotide A2059 in the four compared crystalstructures. Residue K68 is located in vicinity of the phosphate ofnucleotide G2061 (SA G2088, DR G2044) and the sugar-phosphate backboneof A2059 that are in direct contact with linezolid. It has a similarorientation in E70S, SA50S and T70S, but is different from D50S (see,FIG. 13F). The mutation K68Q replaces a long positively charged sidechain by a shorter uncharged one, with no electrostatic interactionswith G2061 and A2059. G69A and T70P point mutations introducehydrophobic residues, which may increase the flexibility of the bindingpocket, thus reducing the efficiency of the binding and explaining howresistance to linezolid is gained in these cases. All available NCBIsequences of rProtein L4 indicate that its residue 68 lysine is highlyconserved; however, in H50S, the respective residue is serine, a shortpolar uncharged residue, which seems to reduce binding efficiency. Thismay be an additional feature of the weaker binding mode of linezolid toH50S.

The L22 hairpin loop is part of the exit tunnel wall point mutations,deletions and insertions in this loop, between residues R80-S108, in SAconfer resistance to erythromycin, synercid and telithromycin althoughits location is too far for direct chemical interactions with the bounddrugs. Interestingly, similar resistance mutations were also observed inadditional bacterial species including some pathogens. Crystal structureof one of these mutants provided the structural basis for thisresistance, showing that the insertion of 3 amino acids (VPR afterR109-D50 numbering) into L22 hairpin loop reshapes the binding region.Thus, the repositioning of the tip of L22 hairpin loop, triggered acascade of structural rearrangements of the rRNA nucleotides thatpropagates to erythromycin binding site, reshaping the tunnel walls in afashion permitting nascent protein progression in the tunnel even in thepresence of erythromycin (see, FIG. 13A). It is conceivable that similarmechanisms play roles in acquiring resistance in SA deletion/insertionmutants.

Multidrug resistance to phenicols, lincosamides, oxazolidinones,pleuromutilins, and streptograminA (PhLOPS_(A)) is facilitated in SA bythe Cfr and RlmN genes that code enzymes that methylate A2503 in twopositions, namely C8 and C2. A2503 is more than 98% conserved throughoutall kingdoms; nevertheless, it can be modified without terminatingprotein biosynthesis. A2503 methylation, which increases its size,interferes with PhLOPS_(A) drugs binding, thus conferring resistance tothem. Similarly, mutation or methylation of A2503G causes linezolidresistance in SA presumably by creating a steric clash with the drug(see, FIG. 13B).

Example 7 Structural Differences and Selectivity Between Eukaryotic andPathogen Ribosome

Selectivity, namely the distinction between bacterial pathogens andeukaryotes, is crucial for clinical usage of antibiotics. Below wehighlight the main differences between the structures of the antibioticsbinding sites in the large ribosomal subunits of the pathogen SA andtheir mates in eukaryotic ribosomes, based on comparisons of SA50Sstructure with the currently available eukaryotic ribosome structures,namely those of S. cerevisiae 80S ribosome [Ben-Shem, A. et al.,Science, 2011, 334(6062), p. 1524-1529] and T. thermophila 60S largeribosomal subunit [Klinge, S. et al., Trends In Biochemical Sciences,2012, 37(5), p. 189-198], as well as on the relevant sequences in humanribosome. Another study compared between the binding pockets ofinhibitors of eukaryotic ribosomes in S. cerevisiae and E. coli [deLoubresse, N. G. et al., Nature, 2014, 513(7519), p. 517].

FIGS. 14A-C present graphic illustrations of regions in the superimposedstructures of S. cerevisiae 60S (PDB ID: 3U5D) (colored in yellow), T.thermophilia 60S (PDB ID: 4A18) (colored in red) and SA50S (colored inteal), showing the sequence and structural variability among eukaryotesand prokaryotes rRNA antibiotics binding pockets and vicinity.

FIG. 14A shows the MLS_(B)K (erythromycin (PDB ID: 3OFR) (shown in red)binding pocket in SA50S and the two eukaryotes structures. FIG. 14Bshows the ketolides binding pocket of telithromycin in SA50S and the twoeukaryotes structures. FIG. 14C shows the PTC binding pocket of tiamulin(PDB ID: 1XBP) (colored in purple) in SA50S and the two eukaryotesstructures.

Macrolides, lincosamides, ketolides and streptogramins_(B) (MLS_(B)K)bind to the protein exit tunnel near to its constriction. Theirselectivity is attributed to the difference in the identity of bindingpocket nucleotide 2058 that binds the desosamine sugar of themacrolides, which is guanine in eukaryotes and adenine in eubacteria. Inaddition, the second shell nucleotides around the macrolide binding siteplay a key role is selectivity. Thus, G2057 and G2056 are base-pairedwith C2611 and C2612 in SA50S. In eukaryotes both 2057 and 2056nucleotides are adenines (see, FIG. 14A), hence their base-pairinginteractions are compromised. These two base-pairs play important rolesin the stabilization of position of G2058. It is worth noting that inD50S and T70S, only one of these base-pairs, namely between C2611 andG2057 is maintained, while the other is replaced by His 4 of L32 thatextents towards the macrolide binding pocket deeper than in SA50S andE70S structures.

Nucleotides C2611 and C2610 belong to the MLS_(B)K binding pocket. Bothare uridines in eukaryotes and the variability in their identity betweenprokaryotes and eukaryotes is also important for pathogen/patientselectivity. Another nucleotide that is related to the selectivity ofSA50S MLS_(B)K binding pocket is C2586 that in SA is located inproximity to the C12-C13 of the erythromycin macrolactone ring and is Uin E. coli and eukaryotes. C1782 and U1781 are additional nucleotidesthat may contribute to SA specificity in the binding pocket of MLS_(B)K.C1782 is U in E. coli and eukaryotes, as well as nucleotide U1781 whichis U in prokaryotes, including SA, but A in eukaryotes (see, FIG. 14A).

Interestingly, in the SA50S and T70S and D50S, nucleotide 790, which isU in all structures except D50S, is pointing into the tunnel, whereas itpoints into the particle's core in the two eukaryotes structures (see,FIG. 14B). In SA50teli, nucleotide U2609 is base-paired with A752 thatis an important determinant of ketolides improved activity; however, asdiscussed above, the drug is not directly interacting with thisbase-pair. Nevertheless, its absence may alter the ketolides bindingpocket thus affecting their inhibitory activity. This base-pair is foundin yeast but not in T. termophila. Additional selectivity determinantsat the MLS_(B)K binding pocket are nucleotide U746, a first shellnucleotide, which is G in eukaryotes, and G753, that is a second shellnucleotide around telithromycin binding pocket, is A in E. coli but U ineukaryotes, whereas residue U754, a third shell nucleotide, is C ineukaryotes (see, FIG. 14B).

The exit tunnel nucleotides that are conserved among prokaryotes versuseukaryotes are A2058, A2057, C2610, C2611, C2612, U1781, U746 and U754.SA sequence specific nucleotides versus eukaryotes and other prokaryotesare C2586, C1782 (both at the exit tunnel) and G753 (second shell totelithromycin binding pocket). Thus, drug selectivity of eubacteriaversus eukaryotes is achieved by eight nucleotides that are located atthe MLS_(B)K binding pocket or in its second to third shells.Interestingly, nucleotides 2586 and 1782 that belong to the MLS_(B)Kbinding site are C in SA but U in other prokaryotes and eukaryotes.These unique identities may play a role in the SA species specific drugresistance mechanisms, and hence may be exploited for the design ofnovel specific antibiotics. Similarly, G753, a second shell nucleotideat the MLS_(B)K binding pocket is G in SA but A in EC and U ineukaryotes might be used for selective distinction between the SA andother ribosomes.

The PTC that is highly conserved in all kingdoms contains flexiblenucleotides, detected also in SA50S. Variations in PTC nucleotidesorientations between the structures of SA50S and other bacterial andeukaryotic ribosomes include the conserved nucleotides, namely A2062,U2504, U2506, U2585 and A2602. A2062, which has a different orientationin SA50S compared to the eukaryotic ribosomes, is found in resistantstrains (see, FIG. 14C). U2504 has been pointed as being at thecrossroad of remote mutations networks that hamper binding of PTCantibiotics. U2585 and U2506 display flexibility upon the pleuromutilinsbinding. U2585 and A2602 are two universally conserved nucleotidesfacilitating the translocation of the 3′-end tRNA A-site to P-site.Among them, U2585, may also be involved in D-amino acid rejection, andin the synergistic action of members of the streptogramin class ofantibacterial agents. Interestingly, the involvement of the PTC secondshell nucleotide 2453 in maintaining the PTC conformation is lessdependent on its identity. Thus, it is A in bacteria and U ineukaryotes. However, although its role in peptide bond formation seemsto be less crucial, it plays a role in drug selectivity at the PTC. Thesecond shell nucleotide C2055 is the only nucleotide around the PTC thatis C in bacteria and A in eukaryotes, thus creating differentinteractions with the first shell nucleotide U2504 in bacteria versuseukaryotes second and third shell nucleotides that are modulating aconserved drug binding site can play roles in selectivity and resistance(see, FIG. 14C). Thus, G2576 and C2055 are both second shell residuesaround the PhLOPS_(A) binding site; G2576 is mutated in resistantstrains while the residue 2055 is C in prokaryotes and A in eukaryotes,thus has role in selectivity.

Example 8 Design of a Novel Ribosomal Ligand

Following is a general procedure for designing a novel ligand for theSA50S subunit, based on the native and complex structures providedherein. A procedure for designing the ligand is based on developing anexpanded pharmacophore and generally involves the following steps:

Providing a set of SA50S complex structures with a diverse set ofligands bound in or near a ribofunctional locus of interest. The set ofatomic coordinates of the diverse bound ligands is used as a trainingset for developing the pharmacophore model.

Superimposing each of the complex structures on the native structure soas to afford a fit between a maximal number of ribosomal atoms indifferent complexes, thereby affording superimposition of the ligands(the members of the training set) in their bound conformation andrelative spatial positions.

Transforming the superimposed ligands into a spatially positioned set ofabstract representations of pharmacophore elements. For example,superimposed phenyl rings from different ligands occupying a relativelysmall space are jointly referred to as an “aromatic ring” pharmacophoreelement at the center of the space. Likewise, hydroxyl groups arereferred to as a “hydrogen-bond donor/acceptor” pharmacophore element.

Identifying the combined pharmacophore having the maximal number ofcontributions (pharmacophore elements) from each of the complexstructures, thereby maximally expanding the pharmacophore.

Designing a rigid molecular skeleton which links spatially all thepharmacophore elements, and designing functional groups on the skeletoncorresponding to each of the pharmacophore elements.

In an exemplary embodiment of the invention, a putative ligand isdesigned as an adduct of moieties which stem from the chemical structureof each of the ligands linezolid, BC-3205 and telithromycin, which areused as a diverse set of ligands for the training set.

According to some embodiments, each of the pre-verified ligands,linezolid, BC-3205 and telithromycin, is individually parsedcomputationally to arbitrary yet chemically viable segments, which canbe used as building blocks of a rationally designed de-novo ligand,according to some embodiments of the present invention.

For example, linezolid can be parsed to the following exemplary andnon-limiting moieties:

wherein each of the wavy lines represent an optional linking positionwhich can be used to link the moiety to a skeleton of an adduct or toanother moiety in the adduct.

Similarly, telithromycin can be parsed to the following exemplary andnon-limiting moieties:

wherein each of the wavy lines represent an optional linking positionwhich can be used to link the moiety to a skeleton of an adduct or toanother moiety in the adduct.

Similarly, BC-3205 can be parsed to the following exemplary andnon-limiting moieties:

wherein each of the wavy lines represent an optional linking positionwhich can be used to link the moiety to a skeleton of an adduct or toanother moiety in the adduct.

It is noted that the parsing of a pre-verified ligand can take otherforms, and each moiety can be further parsed to sub-moieties and soforth.

In the exemplary embodiment presented herein, the superimposition of thecomplex structures over the native structure is based on fittingphosphate atoms of the rRNA chains common to all native and complexstructures.

FIG. 15 presents an illustration of linezolid (green stick model),BC-3205 (blue stick model) and telithromycin (red stick model), as thesethree ligands are positioned in the crystal structure of thecorresponding complex with SA50S, wherein each complex structures issuperimposed on the native SA50S crystal structure, and further presentsthe molecular surface of the combined ligand structures illustrated as awire mesh encasing the three ligands, wherein the coloring of meshcorresponds to the color of the ligand which contributes to themolecular surface at the corresponding region thereof.

As can be seen in FIG. 15, linezolid and BC-3205 occupy a similarbinding site commonly referred to as the PTC, while telithromycinoccupies a proximal binding site commonly referred to as the polypeptidetunnel opening. As can further be seen in FIG. 15, the relatively shortdistance between the atoms of BC-3205 and telithromycin (about 2.5]) canreadily be bridged by a linking moiety in the form of an linking, forexample, the isopropyl moiety of BC-3205 (a sub-moiety of the(R)-2-amino-N,N,3-trimethylbutanamide illustrated hereinabove) to thepyridine moiety of telithromycin. Alternatively, the deoxy-sugar moietyof telithromycin((2R,3R,4S,6R)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2,3-diol)can be linked by a linking moiety to the vinyl moiety of BC-3205 (asub-moiety of the (3S,4R)-4-methylhex-5-en-3-ol illustratedhereinabove).

Hence, an adduct-type ligand is designed to substantially occupy thespace which is at least partially occupied by all three ligands, namelyat least a portion of the space delineated in the wire mesh illustrationin FIG. 15, and exhibit moieties which belong to the expandedpharmacophore corresponding to the combined structures of all threeligands, such as for a non-limiting example, the morpholine and theN-methylacetamide moieties of linezolid, the cyclopentanone and the2-amino-3-methyl-1-(piperidin-1-yl)butan-1-one moieties of BC-3205, andthe 3-(1H-imidazol-4-yl)pyridine and the oxazolidin-2-one moieties oftelithromycin. The moieties are linked by molecular skeleton and linkingmoieties which are designed to exhibit the moieties in the relativepositions and conformation corresponding to the positions andconformations of the corresponding moieties as they appear in the boundform of the corresponding ligand in each of the complex crystalstructure. An exemplary skeleton for an exemplary de-novo designedligand, according to some embodiments of the present invention, can be amoiety that mimics or duplicates the macrolide ring (the largemacrocyclic lactone) of telithromycin, or a moiety that mimics orduplicates the bicyclo[5.3.1]undecane or the tricyclic mutilin core ofBC-3205. Alternatively, the skeleton can be any alkyl, heteroalkyl,alicyclic, hetero-alicyclic moiety, aryl and/or heteroaryl moiety, andany combination thereof.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

Example 9

The complex crystal structure of SA50S-lefamulin (SA50Slef) Lefamulin(BC-3781), developed by Nabriva Therapeutics, Vienna, Austria, is asemi-synthetic pleuromutilin compound that is highly active againstpathogens that are commonly associated with community-acquired bacterialpneumonia (CABP), including multi-drug resistant S. pneumoniae, S.aureus, and M. pneumonia.

Crystal Structure of SA50S-Lefamulin Complex:

The complex structure of the antibiotic drug lefamulin bound to SA50S,SA50S crystals, obtained essentially as described hereinabove weresoaked in a solution containing 22.8 μg/ml lefamulin in a stabilizationsolution for 6 hours prior to flash freezing and data collection.

The crystal structure of the large ribosomal subunit from S. aureuscomplexed with lefamulin (SA50Slef; PDB ID: 5HL7) was obtained andcharacterized as summarized in Table 6 below (in parentheses are thevalues for the highest resolution shells of 3.61-3.55).

TABLE 6 Subject SA50Slef Space group P6₅22 a = b [Å] 282.1 c [Å] 875.3α, β, γ [°] 90, 90, 120 Complex with lefamulin (BC-3781) X-ray sourceID23-1 (ESRF) Wavelength [Å] 0.972 Number of crystals 12 Resolution [Å]  50-3.55 (3.61-3.55) Unique reflections 236087 Observed reflections2606786 Redundancy 11.0 (9.2)  Completeness [%] 95.7 (94.6) <I>/<σ>  9.0(1.71) R-merge [%] 20.9 (99.4) Refinement R-factor [%] 18.69 R-free (5%)[%] 22.66 RMSD bonds [Å] 0.010 RMSD angles [°] 1.610

FIG. 16 presents a graphic illustration of an overlay of lefamulin inthe pleouromutilin binding site, as elucidated from the complexstructures SA50Slef PDB ID: 5HL7 (orange), D50S-retapamulin (cyan; PDBID: 2OGO) and D50S-SB280080 (lemon; PDB ID: 2OGN).

FIG. 17 presents a graphic illustration of an overlay of the PTC innative SA50S (teal) and in SA50Slef complex PDB ID: 5HL7 (orange),revealing the movements of nucleotide U2585 in the bound vs. nativestructure.

FIG. 18 presents a graphic illustration of an overlay of the PTC incomplex structure SA50SBC-3205 (magenta) and in complex structureSA50Slef (orange).

As can be seen in FIG. 16, in the SA50Slef complex crystal structure,lefamulin was found to be bound at the PTC, so that its tricyclicmutilin core is blocking the A-site, and its C14 extension is pointinginto the P-site, thus perturbing A- and P-site tRNA accommodation, aswas found in the ribosome-pleuromutilin complexes with D50S and SA50S.

As can further be seen in FIGS. 16-18, as all pleuromutilins are assumedto bind to the same pocket, a hydrogen bond is formed between the drug'sacetyl carbonyl and the NH₂ of G2061. In the SA50Slef complex, theconformation of the flexible nucleotide U2585 is different from that ofthe unbound SA50S and of the SA50SBC-3205 complex. Interactions with therRNA nucleotides C2063, U2506, A2503, U2504, G2505, A2453, C2452, A2425and C2424 are either hydrophobic or based on van der Waals forces.

FIG. 19 presents a graphic illustration of the ribosomal binding pocketof lefamulin, as seen in the complex crystal structure SA50Slef, showingthat the ligand is held within the PTC by a net of hydrogen bonds withthe 23S rRNA, wherein the U-U interactions between U2585 and U2506stabilizes the lefamulin binding pocket (the electron density oflefamulin is weighted 2F_(o)-F_(c) contoured at 1.0 σ).

As can be seen in FIG. 19, in the SA50Slef complex, U2585 conformationalrange is reduced because of a stacking interaction with the C14extension of lefamulin. This interaction is stabilized by aU-U-4-carbonyl N3 symmetric interactions between U2585 and U2506, whichis shifted towards the binding pocket. Another hydrogen bond is formedbetween NH2 group of the lefamulin C14 extension and the 02 of A2062ribose.

FIGS. 20A-B present the results of in vitro transcription-translationcell-free inhibition assays of bacterial protein synthesis, wherein theinhibitory effect on protein expression in S. aureus system of BC-3205is presented in FIG. 20A and of lefamulin is presented in FIG. 20B. Theactivity of the reporter protein (luciferase) in the presence of variousconcentrations of BC-3205 and lefamulin is shown as arbitrary unit ofluminescence [a.u.]. The IC50 values calculated by the plotted datashowed better inhibition of lefamulin than BC-3205 on protein synthesis.

As can be reckoned from the results, lefamulin has a single additionalinteraction with the PTC 23S rRNA nucleotides compared to BC-3205 (withA2062) and its U2585 and U2506 U-U interactions stabilize the lefamulinbinding pocket. Compared to other pleuromutilins, it makes twoadditional hydrogen bonds. This might be the reason for lefamulin priorpotency over BC-3205 in S. aureus cell free in vitrotranscription-translation assay; IC50=0.09 μg/ml of lefamulin comparingto IC₅₀ 0.42 μg/ml of BC-3205 (see, FIGS. 20A-B).

Following the rationale for novel species-specific protein synthesisinhibitors, such as presented in Example 8 hereinabove, the SA50Slefcomplex structure may contribute structural data pertaining to spatialpositioning of moieties in the lefamulin molecule, that interact withvarious ribosomal moieties, and particularly lefamulin moieties thatinteract with ribosomal moieties that differ structurally and chemicallyacross species. One non-limiting example of such a moiety is2-((4-amino-2-hydroxycyclohexyl)thio)acetyl:

Example 10 The Complex Crystal Structure of SA50S with Lincosamides

Lincosamides are known to inhibit the peptidyl transferase reactiontaking place in the ribosome during transcription-translation.Lincomycin, a member of the lincosamides family of antibacterial drugs,is a naturally occurring compound that is produced by the bacteriaStreptomyces lincolnensis. It is a narrow-spectrum antibiotic agent andtargets primarily Gram-positive bacteria, including pathogenicStreptococci, Staphylococci, Mycoplasma, anaerobic bacteria, mostanaerobic bacteria such as Bacteroides fragilis, and some protozoa.Lincomycin is used to treat severe bacterial infections in people whocannot tolerate penicillin-type antibiotics. It shows weak activityagainst most Gram-negative bacteria.

Clindamycin, a semi-synthetic lincosamide antibiotic agent. Lincomycinstructure is similar to clindamycin, except for its 7-hydroxy group,which is a chlorine atom in clindamycin. Clindamycin has largelyreplaced lincomycin due to an improved side effect profile, and due toits higher potency against Gram-negative bacteria. This is in part dueto the higher lipid solubility of clindamycin that enables it topermeate the outer membrane of the target bacteria.

Shortly after the introduction of erythromycin in therapy in the 1950s,resistance to the drug was observed in bacterial pathogens. Moredisquieting was the observation that Staphylococcus aureuserythromycin-resistant strains were cross-resistant not only to allother macrolides but also to the chemically unrelated lincosamide andstreptogramin B drugs. This phenomenon was termed themacrolide-lincosamide-streptogramin B (MLS_(B)) antibiotic resistancephenotype and was found to be caused by to expression of amethyltransferase enzyme, ermC. This enzyme methylates 23S rRNA at theN-6 position of adenosine A2058 (Escherichia coli numbering throughout),which is a pivotal nucleotide for the binding of MLS_(B) antibiotics.Later, an additional methyltransferase enzyme, encoded by the Cfr gene,was found to cause multidrug resistance in S. aureus to phenicols,lincosamides, oxazolidinones, pleuromutilins, and streptogramin A(PhLOPSA) by methylation of the C8 position of the 23S rRNA nucleotideA2503. Even though resistance in bacteria with multiple rrn operons,such as Staphylococcus species, is generally conferred by rRNAmodifications such as these methylations, several mutations are rRNA andrProteins were identified in S. aureus.

The A2058G, A2058U mutations in the 23S rRNA were associated withMLS_(B) resistance, similar to those already reported for otherorganisms. A2059G mutation is associated with macrolide-lincosamideresistance; however the insusceptibility to lincosamides seemed to bemoderate as previously reported for Helicobacter pylori andStreptococcus pneumoniae. Cross-resistance to chloramphenicol,linezolid, and streptogramin A, of which the binding pockets partiallyoverlap, is associated with the second shell nucleotide G2576U mutation.In addition, cross-resistance of S. aureus to chloramphenicol andlinezolid may be caused by G2505A and U2500A mutations. The mutationK68Q in the rProtein uL4 in S. aureus causes resistance tochloramphenicol, linezolid, pleuromutilins, and tedizolid. This residue(68) has been identified in the S. aureus crystal structure providedherewith, and found to be in proximity to the mentioned above A2058 andA2059.

To obtain SA50S-lincomycin complex (SA50Slinc), SA50S crystals, providedas described hereinabove, were soaked in solutions containing 22 μg/mllincomycin in the stabilization solution for 6 hours prior to flashfreezing and data collection. The crystal structures of the largeribosomal subunit from S. aureus, complexed with lincomycin (SA50Slinc;PDB ID: 5HKV) was determined according to the procedure described above.The crystallographic results are summarized in Table 7 below (inparentheses are the values for the highest resolution shells of 5HKV(3.70-3.64).

TABLE 7 Subject SA50Slinc Space group P6₅22 A = b [Å] 280.8 c [Å] 873.5α, β, γ [°] 90, 90, 120 Complex with lincomycin X-ray source ID29,ID23-1 Wavelength [Å] 0.971 Number of crystals 30 Resolution [Å]  50-3.64 (3.70-3.64) Unique reflections 2230088 Observed reflections223602 Redundancy 10.0 (4.4)  Completeness [%] 98.0 (78.1) <I>/<σ> 9.08(1.37) R-merge [%] 23.7 (97.2) Refinement R-factor [%] 18.74 R-free (5%)[%] 23.40 RMSD bonds [Å] 0.009 RMSD angles [°] 1.438

FIG. 21 presents a graphic illustration of the electron density map(weighted 2Fo-Fc contoured at 1.0 σ) attributed to a molecule oflincomycin as seen in the crystal structure of the antibiotic agentcomplexed with SA50S (PDB ID: 5HKV).

FIG. 22 presents a graphic illustration showing a structuralsuperposition of the two lincosamides in their common binding site,wherein the structure of the bound lincomycin is derived from SA50SlincPDB ID: 5HKV (presented in pink) and structure of the bound clindamycinis derived independently from PDB ID: 1JZX disclosing D.radiodurans-lincomycin complex (D50S-CLY, presented in grey), PDB ID:1YJN disclosing H. marismortui-lincomycin complex (H50S-CLY, presentedin sky-blue), and PDB: ID 3OFZ disclosing E. coli-lincomycin complex(E70-CLY, presented in green).

As can be seen in FIG. 22, the pyrrolidinyl propyl group of the twolincosamides is positioned at the PTC and interferes with A-site tRNApositioning while their lincosamine moieties point towards the nascentchain exit tunnel.

FIGS. 23A-B present a graphical illustration of the binding pocket oflincomycin in SA50S, wherein FIG. 23A shows lincomycin (presented inpink) interacts with the PTC A-site by numerous hydrogens bond (dashedline) with the 23S rRNA (presented in grey), and FIG. 23B is a 90degrees horizontal rotated view of FIG. 23A.

The available crystal structures indicate that the two lincosamidesexhibit four hydrogen bonds with the 23S rRNA nucleotides surroundings4): (1) O2 group of the lincosamine forms a hydrogen bond with N4 ofC2611; (2) O3 group of the lincosamine forms a hydrogen bond with N6 ofA2058; (3) O4 group of the lincosamine forms hydrogen bond with O2 ofA2503's sugar; and (4) The bridging amine (NH) of lincomycin createshydrogen bond to the ribose O4 of G2505. The lincomycin's andclindamycin's (H50S-CLY and E70S-CLY structures) O2, O3 and O4 groupsare also involved in hydrogen bonding with N1 of A2058, N6 of A2059 andOP1 of G2505, respectively. Other interactions of lincomycin with therRNA nucleotides U2504, C2452, A2451, G2061 and U2506 are eitherhydrophobic interactions or van der Waals force interactions.

FIG. 24 present a graphical illustration of a structural superpositionof the PTC in native SA50S (presented in teal) and in SA50S-lincomycincomplex (presented in pink), showing a difference in the position ofnucleotide A2062 in the SA50Slinc towards the spermidine (SPD) comparedto the native structure.

Comparisons of the SA50Slinc, D50S-CLY, H50S-CLY and E70S-CLY complexstructures show that nucleotide A2062 has different orientations. In S.aureus, E. coli and D. radiodurans the orientations are only slightlydifferent, whereas in H. marismortui this nucleotide is shifted towardsclindamycin, so that N6 of A2062 forms a hydrogen bond with the carbonylof the clindamycin. In SA50Slinc electron density map an additionalelectron density was observed between the lincomycin and nucleotideA2062 that can accommodate a molecule of spermidine (an additive of thecrystallization conditions). This spermidine interacts with the7-hydroxy group of the lincomycin and with A2062 thus stabilizes A2062in its place which is slightly different from the non-bound structure(SA50S). The interaction with A2062 does not occur with clindamycinsince it contains a chlorine atom instead of the 7-hydroxy group.

U2504 and U2506 are in a similar position in SA50Slinc and E70S-CLY butdiffer from D50S-CLY and H50S-CLY. A2503 in D50S-CLY is flipped by 180degrees from its position in the other structures. In SA50Slinc U2585nucleobase could not be placed in the electron density map due to itsflexibility while in the SA50S complexed with clindamycin and in SA50Sit has a slightly different orientation (see, FIGS. 23A-B).

In D50S-CLY the clindamycin is positioned somewhat different, so thatits pyrrodinyl propyl tail is pointing 102 degrees away from itsposition in the other available structures.

As can be concluded from the data and structural analysis of SA50Slincthe other available structures of 50S-lincomycin complex, lincosaminesbind at the same binding pocket in the large ribosomal subunit. Itappears that in the studied organisms, the lincosamide sugar has similarH-bond interactions with the 23S nucleotides. This sugar moiety may bethe main determinant for targeting the lincosamines to the ribosome. Thestructural data presented herein shows that by replacing the moiety thatis attached to the sugar moiety, binding and inhibition can still beachieved mainly through this moiety.

It is also seen that lincomycin complex with SA50S accommodates aspermidine in the binding pocket. This spermidine, a polyamine,interacts with the 7-hydroxyl of the lincomycin and with A2062. Underphysiological conditions this spermidine can be replaced by an ion,which tightens the lincomycin pocket. In the complex H50S-CLY A2062 N6forms hydrogen bond with the carbonyl, but in the D50S-CLY and E70S-CLYthere is no hydrogen bond between this nucleotide and clindamycin. Itseems that the interaction with A2062 improves the drug bindingproperties as lincomycin has a super low IC₅₀ against S. aureus ribosomecompared to E. coli ones (available data no shown).

The Crystallographic data indicate that the interactions of the4-propyl-2-pyrrolidinyl ring of the lincosamide antibiotics lincomycinand clindamaycin with the 23S nucleotides are based essentially on vander Waals forces. This data may indicate that the contribution of the4-propyl-2-pyrrolidinyl moiety is small, yet, this moiety affects thepharmacokinetic properties of lincosamides and its overall proteintranslation inhibition properties.

As lincosamines bind at the tRNA A-site and at the entrance to the exittunnel so that the lincosamine sugar moiety overlaps with the siteoccupied by the desosamine sugar of macrolides, the cross-resistancebetween lincosamines and the macrolides may be explained thereby. Fromthe comparison with the macrolide it may be rationalized why the sugarmoiety of the lincosamides alone does not inhibit protein synthesis(available data no shown).

Following the rationale for novel species-specific protein synthesisinhibitors, such as presented in Example 8 hereinabove, the SA50Slefcomplex structure may contribute structural data pertaining to spatialpositioning of moieties in the lincomycin molecule, that interact withvarious ribosomal moieties, and particularly lincomycin moieties thatinteract with ribosomal moieties that differ structurally and chemicallyacross species. Non-limiting example of such moieties includelincosamine and 4-propyl-2-pyrrolidinyl moieties.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting. In addition, any priority document(s) of this applicationis/are hereby incorporated herein by reference in its/their entirety.

What is claimed is:
 1. A method producing a ligand having an affinity toa binding site of a large ribosomal subunit of a pathogenic bacterium,the method comprising: (a) obtaining positioning data indicative ofatomic coordinates of at least one binding site determined from anelectron density map having a resolution of at least 4 Å calculated fromX-rays diffraction data obtained using at least one of acomposition-of-matter that comprises a crystallized Staphylococcusaureus 50S large ribosomal subunit, wherein the crystallized 50S largeribosomal subunit effectively diffracts X-rays for calculating anelectron density map and determination of atomic coordinates to aresolution of at least 4 Å and forms in a hexagonal space group withunit cell dimensions of a=279.6±10 Å, b=279.6±10 Å, c=872.7±10 Å, α=90,β=90, γ=120; (b) calculating a molecular surface of the binding site;(c) computationally constructing a model of a chemically feasible ligandhaving a molecular surface that match the molecular surface of thebinding site; and synthesizing the ligand based on said model.
 2. Themethod of claim 1, wherein said Staphylococcus aureus is capable ofdeveloping a resistance to an antibacterial agent.
 3. The method ofclaim 2, wherein said Staphylococcus aureus is selected from the groupconsisting of a methicillin-resistant Staphylococcus aureus (MRSA), anoxacillin-resistant Staphylococcus aureus (ORSA), a vancomycin-resistantStaphylococcus aureus (VRSA) and a vancomycin intermediateStaphylococcus aureus (VISA).
 4. The method of claim 2, characterized bythe atomic coordinates deposited at the Protein Data Bank underaccession number PDB ID: 4WCE.
 5. The method of claim 1, wherein aligand is bound to said large ribosomal subunit to form a crystallizedcomplex of the subunit and said ligand.
 6. The method of claim 5,wherein said ligand is selected from the group consisting of linezolid,BC-3205, telithromycin, lefamulin and lincomycin.
 7. The method of claim6, wherein said ligand is linezolid and the composition is characterizedby the atomic coordinates deposited at the Protein Data Bank underaccession number PDB ID: 4WFA.
 8. The method of claim 6, wherein saidligand is BC-3205 and the composition is characterized by the atomiccoordinates deposited at the Protein Data Bank under accession numberPDB ID: 4WFB.
 9. The method of claim 6, wherein said ligand istelithromycin and lincomycin and the composition is characterized by theatomic coordinates deposited at the Protein Data Bank under accessionnumber PDB ID: 4WF9.
 10. The method of claim 6, wherein said ligand islefamulin and the composition is characterized by the atomic coordinatesdeposited at the Protein Data Bank under accession number PDB ID: 5HL7.11. The method of claim 6, wherein said ligand is lincomycin and thecomposition is characterized by the atomic coordinates deposited at theProtein Data Bank under accession number PDB ID: 5HKV.
 12. The method ofclaim 1, further comprising, prior to step (c): computationallyconstructing a library of structures of chemically feasible ligandshaving a molecular surface that matches the molecular surface of thebinding site.
 13. The method of claim 12, further comprising: (d)computationally determining a matching score for each of said ligands;and (e) based on said matching score selecting at least one putativeligand having the desired affinity to the binding site of the largeribosomal subunit of a pathogenic bacterium.
 14. The method of claim 13,further comprising, prior to step (d), adding to said library aplurality of structures of chemically feasible variants of pre-existingligands.
 15. The method of claim 1, further comprising, prior to step(c), calculating a molecular surface of at least a portion of thebinding site of a large ribosomal subunit of a different organism. 16.The method of claim 15, wherein said different organism is selected fromthe group consisting of a host of the pathogenic bacterium and a benignmicroorganism.
 17. The method of claim 16, wherein step (c) comprisescomputationally constructing a chemically feasible ligand having amolecular surface that matches the molecular surface of the binding siteof the large ribosomal subunit of a pathogenic bacterium, and mismatchesat least one feature in said molecular surface of the binding site insaid of a large ribosomal subunit of said different organism.
 18. Themethod of claim 1, wherein the binding site is selected from the groupconsisting of a inter-subunit interface, a peptidyl transferase site, aGTPase center, an mRNA binding site, an A-site, a P-site, an E-site, apolypeptide exit tunnel, a translation initiation factor (IF1) bindingsite, a translation initiation factor (IF2) binding site, a translationinitiation factor (IF3) binding site, an elongation factor G (EF-G)binding site, elongation factor Tu (EF-Tu) binding site, hibernationfactor HPF binding site, hibernation factor RMF binding site,hibernation factor YfiA binding site, a GTP binding site and a ricinbinding site.
 19. The method of claim 1, wherein the ligand has amolecular weight of less than 1,500 g/mol.